Genotyping methods using genomic sequence of the 5-lipoxygenase-activating protein (FLAP) and polymorphic markers thereof

ABSTRACT

The invention concerns the genomic sequence of the FLAP gene. The invention also concerns biallelic markers of a FLAP gene and the association established between these markers and diseases involving the leukotriene pathway such as asthma. The invention provides means to determine the predisposition of individuals to diseases involving the leukotriene pathway as well as means for the diagnosis of such diseases and for the prognosis/detection of an eventual treatment response to agents acting on the leukotriene pathway.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.11/512,789, filed Aug. 29, 2006, which is a divisional of U.S.application Ser. No. 10/359,512, filed Feb. 5, 2003, now U.S. Pat. No.7,118,869, which is a divisional of U.S. application Ser. No.09/292,542, filed Apr. 15, 1999, now U.S. Pat. No. 6,531,279, whichclaims priority from U.S. Provisional Application Ser. No. 60/081,893,filed Apr. 15, 1998; U.S. Provisional Application Ser. No. 60/091,314,filed Jun. 30, 1998; and U.S. Provisional Application Ser. No.60/123,406, filed Mar. 8, 1999, all of which are hereby incorporated byreference herein in their entireties, including any figures, tables,nucleic acid sequences, amino acid sequences, or drawings.

FIELD OF THE INVENTION

The invention concerns the genomic sequence of the FLAP gene. Theinvention also concerns biallelic markers of a FLAP gene and theassociation established between these markers and diseases involving theleukotriene pathway such as asthma. The invention provides means todetermine the predisposition of individuals to diseases involving theleukotriene pathway as well as means for the diagnosis of such diseasesand for the prognosis/detection of an eventual treatment response toagents acting on the leukotriene pathway.

BACKGROUND OF THE INVENTION

The progression of inflammatory diseases in which the synthesis ofleukotrienes plays an active role, such as asthma and arthritis,constitutes a major health problem in Western societies.

For example, the prevalence of asthma in Occidental countries has risensteadily over the last century, affecting about 10% of the population.In 1994, it afflicted more than 14 million people in the United Statesalone (including 4.8 million (6.9%) less than 18 year of age) whereasonly 8 million people suffered from the same disease in 1982. It claimsmore than 5000 lives each year (including 342 deaths among persons agedless than 25 in 1993). Asthma affects one child in seven in GreatBritain, and in the United States, it causes one-third of pediatricemergency-room visits. It is the most frequent chronic disease inchildhood.

Bronchial asthma is a multifactoral syndrome rather than a singledisease, defined as airway obstruction and characterized by inflammatorychanges in the airways and bronchial hyper-responsiveness. Stimuli whichcause the actual asthma attacks include allergens (in sensitizedindividuals), exercise (in which one stimulus may be cold air),respiratory infections and atmospheric pollutants such as sulphurdioxide. The asthmatic subject has intermittent attacks of dyspnea(difficulty in breathing out), wheezing, and cough that can belife-threatening or even fatal.

The manifestation of asthma probably involves both genetic andenvironmental factors, and in most subjects the asthmatic attackconsists of two phases which illustrate the pathophysiology of thecondition:

-   -   an immediate phase, consisting mainly of bronchospasms due to        spasms of the bronchial smooth muscle; the cells involved are        mast cells releasing histamine, but also eosinophils,        macrophages and platelets releasing leukotrienes,        prostaglandins, and platelet-activating factor; these spasmogens        added to chemotaxins and chemokines attract leukocytes into the        area, setting the stage for the delayed phase,    -   a later phase consisting of a special type of inflammation        comprising vasodilatation, oedema, mucus secretion and        bronchospasm; it is caused by inflammatory mediators released        from activated cytokine-releasing T cells and eosinophils, and,        possibly, neuropeptides released by axon reflexes; these        mediators cause damage and loss of bronchial epithelium.

The strongest identifiable predisposing factor for developing asthma isatopy, the predisposition for the development of an IgE-mediatedresponse to common aeroallergens. When IgE binds to the IgE receptors onthe cells, the system becomes primed so that subsequent re-exposure tothe relevant allergen will cause an asthmatic attack. Most asthma cases(95%) are associated with atopy.

Further to their above-mentioned role in asthma, leukotrienes are moregenerally involved in host defense reactions and play an important rolein immediate hypersensitivity as well as in inflammatory diseases otherthan asthma such as inflammatory bowel disease, psoriasis and arthritis.

The Leukotriene Pathway

Leukotrienes are products of the Lipoxygenase pathways. Lipoxygenasesare soluble enzymes located in the cytosol and are found in lung,platelets, mast cells, and white blood cells. The main enzyme in thisgroup is 5-Lipoxygenase which is the first enzyme in the biosynthesis ofleukotrienes.

The first step in leukotriene biosynthesis is the release of arachidonicacid from membrane phospholipids upon cell stimulation (for example, byimmune complexes and calcium ionophores). Arachidonic acid is thenconverted into leukotrienes A4 by a 5-Lipoxygenase (5-LO) whichtranslocates to the cell membrane where it becomes associated to aprotein called “five-Lipoxygenase activating protein” (FLAP), which isnecessary for leukotriene synthesis in intact cells. 5-LO also hasleukotriene A4 hydrolase activity.

Leukotriene A4 (LTA4), an unstable epoxide intermediate, is thenhydrolyzed into leukotriene B4 (LTA4-hydrolase activity) or conjugatedwith glutathione to yield leukotriene C4 (LTC4-synthase activity) andits metabolites, leukotriene D4 and leukotriene E4. LTB4 is producedmainly by neutrophils, while cystinyl-leukotrienes (LTC4, LTD4, andLTE4) are mainly produced by eosinophils, mast cells, basophils, andmacrophages.

LTB4 is a powerful chemotactic agent for both neutrophils andmacrophages. On neutrophils, it also causes up-regulation of membraneadhesion molecules and increases the production of toxic oxygen productsand the release of granule enzymes. On macrophages and lymphocytes, itstimulates proliferation and cytokine release. Thus LTB4 is an importantmediator in all types of inflammations.

Cystinyl-leukotrienes act on the respiratory and cardiovascular systems.In the respiratory system, they are potent spasmogens causing acontraction of bronchiolar muscle and an increase in mucus secretion. Inthe cardiovascular system, they cause vasodilatation in most vessels,but they also act as coronary vasoconstrictors. Thecystinyl-leukotrienes are of particular importance in asthma.

FLAP (5-Lipoxygenase-Activating Protein)

FLAP is a 18-kD membrane-bound polypeptide which specifically bindsarachidonic acid and activates 5-LO by acting as an arachidonic acidtransfer protein. The FLAP gene spans greater than 31 kb and consists offive small exons and four large exons (See GenBank 182657, Kennedy etal. 1991 incorporated herein by reference, Genbank M60470 for exon 1,Genbank M63259 for exon 2, Genbank M63260 for exon 3, Genbank M63261 forexon 4, and Genbank M6322 for exon 5).

The nuclear envelope is the intracellular site at which 5-LO and FLAPact to metabolize arachidonic acid, and ionophore activation ofneutrophils and monocytes results in the translocation of 5-LO from anonsedimentable location to the nuclear envelope. Inhibitors of FLAPfunction prevent translocation of 5-LO from cytosol to the membrane andinhibit 5-LO activation. They are thus interesting anti-inflammatorydrug candidates. Indeed, antagonists of FLAP can attenuateallergen-induced bronchoconstrictor responses which supports animportant role for cystinyl leukotrienes in mediating these asthmaticresponses.

Pharmacogenomics

To assess the origins of individual variations in disease susceptibilityor drug response, pharmacogenomics uses the genomic technologies toidentify polymorphisms within genes that are part of biological pathwaysinvolved in disease susceptibility, etiology, and development, or morespecifically in drug response pathways responsible for a drug'sefficacy, tolerance, or toxicity, including but not limited to drugmetabolism cascades.

In this regard, the inflammatory phenomena which are involved innumerous diseases present a high relevance to pharmacogenomics bothbecause they are at the core of many widespread serious diseases, andbecause targeting inflammation pathways to design new efficient drugsincludes numerous risks of potentiating serious side-effects. Theleukotriene pathway is particularly interesting since its products arepowerful inflammatory molecules.

The vast majority of common diseases, such as cancer, hypertension anddiabetes, are polygenic (involving several genes). In addition, thesediseases are modulated by environmental factors such as pollutants,chemicals and diet. This is why many diseases are called multifactoral;they result from a synergistic combination of factors, both genetic andenvironmental.

For example, in addition to the evidenced impact of environmentalfactors on the development of asthma, patterns of clustering andsegregation analyzes in asthmatic families have suggested a geneticcomponent to asthma. However, the lack of a defined and specific asthmaphenotype is proving to be a major hurdle for reliably detectingasthma-associated genes.

Asthma is usually diagnosed through clinical examination and biologicaltesting. The non-specific bronchial hyper-responsiveness thataccompanies asthma is measured by the variation of airflow triggered ina patient by the administration of a bronchoconstrictor such ashistamine or methacholine. Atopy is detected by skin prick tests thatmeasure serum IgE titers. Standard symptom questionnaires are alsocommonly used to detect symptoms characteristic of, but not unique to,asthma (like nocturnal wheeze and breathlessness).

However, there is no straightforward physiological or biological bloodtest for the asthmatic state. Despite advances in understanding thepathophysiology of asthma and its development, evidence suggests thatthe prevalence of the asthmatic state and the severity of asthma attacksis underestimated. As a result, adequate asthma treatment is oftendelayed, thereby allowing the inflammation process to better establishitself. Thus, there is a need for an efficient and reliable asthmadiagnostic test.

Drug efficacy and toxicity may also be considered as multifactoraltraits that involve genetic components in much the same way as complexdiseases. In this respect, there are three main categories of genes thatmay theoretically be expected to be associated with drug response,namely genes linked with the targeted disease, genes related to thedrug's mode of action, and genes involved in the drug's metabolism.

The primary goal of pharmacogenomics in the study of asthma is to lookfor genes that are related to drug response. It can first provide toolsto refine the design of drug development by decreasing the incidence ofadverse events in drug tolerance studies, by better defining patientsubpopulations of responders and non-responders in efficacy studies and,by combining the results obtained therefrom, to further allow for betterindividualized drug usage based on efficacy/tolerance prognosis.

Pharmacogenomics can also provide tools to identify new targets for drugdesign and to optimize the use of already existing drugs, in order toeither increase their response rate and/or exclude non-responders fromparticular treatments, or decrease undesirable side-effects and/orexclude from corresponding treatment patients with significant risk ofundesirable side-effects.

For this second application of pharmacogenomics, the leukotrienespathway is also useful because many anti-asthmatic and anti-inflammatoryagents which act through the leukotrienes pathway are under development,most of which show some incidence of severe side-effects.

For example, there are two major categories of anti-asthma drugs:bronchodilators and anti-inflammatory agents. Bronchodilators areeffective in reversing the bronchospasm of the immediate phase of thedisease. Drugs used as bronchodilators include the β₂-adrenoceptoragonists (dilating the bronchi by a direct action on the smooth muscle,e.g. salbutamol), the xanthines (e.g. theophylline) and themuscarinic-receptor antagonists (e.g. ipratropium bromide). Theserepresent the short term attack symptomatic treatment.

Anti-inflammatory agents are effective in inhibiting or preventing theproduction of inflammatory components in both asthma phases. Theyinclude glucocorticoids, sodium cromoglicate and histamine H1-receptorantagonists. These agents represent the current long term treatment ofthe asthmatic state.

However, none of these currently used anti-asthmatic drugs is completelysatisfactory as none actually “cures” all patients with the disease.Glucocorticoids are the most interesting active compounds in this regardbut they have potentially serious unwanted side-effects (oropharyngealcandidacies, dysphonia and osteoporosis for inhaled glucocorticoids, andmood disturbances, increased appetite and loss of glucose control indiabetics for systemic glucocorticoids).

In recent years, more effective and selective leukotriene biosynthesisinhibitors (e.g., 5-LO and FLAP-binding inhibitors) have been developedand used as novel therapies for bronchial asthma and other inflammatorydisorders. For example, Zileuton (Zyflo®), an inhibitor of 5-LOcommercialized by Abbott Laboratories (Abbott Park, Ill.), has beenshown to improve airway function and to reduce asthma-related symptoms.

Unfortunately, undesirable side-effects such as acute exacerbation ofasthma, dyspepsia and elevated liver enzymes have been reported inclinical trials for Zileuton. There is also concern about druginteractions with hepatically cleared medicaments.

Thus, in addition to the need for the development of an efficient andreliable asthma diagnostic test, there is also a need to develop moreeffective and better targeted therapeutic strategies acting on theleukotrienes pathway with reduced side-effects and low toxicity for theuser. One way to achieve this in the relative short term would bethrough the use of pharmacogenomics results, to better define the use ofexisting drugs or drug candidates in order to enhance the benefit/riskratio on target subpopulations of patients.

SUMMARY OF THE INVENTION

The present invention stems from the isolation and characterization ofthe whole genomic sequence of the FLAP gene including its regulatoryregions. Oligonucleotide probes and primers hybridizing specificallywith a genomic sequence of FLAP are also part of the invention. Afurther object of the invention consists of recombinant vectorscomprising any of the nucleic acid sequences described in the presentinvention, and in particular recombinant vectors comprising theregulatory region of FLAP or a sequence encoding the FLAP enzyme, aswell as cell hosts comprising said nucleic acid sequences or recombinantvectors. The invention also encompasses methods of screening ofmolecules which modulate or inhibit the expression of the FLAP gene. Theinvention also comprises a new allelic variant of the FLAP protein.

The invention is also directed to biallelic markers that are locatedwithin the FLAP genomic sequence, these biallelic markers representinguseful tools in order to identify a statistically significantassociation between specific alleles of the FLAP gene and diseasesinvolving the leukotriene pathway such as inflammatory diseases, orbetween specific alleles of FLAP gene and either side-effects resultingfrom the administration of agents acting on the leukotriene pathway,preferably Zileuton, or a beneficial response to treatment with agentsacting on the leukotriene pathway. These associations are within thescope of the invention.

More particularly, the present invention stems from the identificationof genetic associations between alleles of biallelic markers of the FLAPgene and asthma, as confirmed and characterized in a panel of humansubjects.

Methods and products are provided for the molecular detection of agenetic susceptibility in humans to diseases involving the leukotrienepathway such as inflammatory diseases and comprising, among others,asthma, arthritis, psoriasis and inflammatory bowel disease. They can beused for diagnosis, staging, prognosis, and monitoring of such diseases,which processes can be further included within treatment approaches. Theinvention also provides for the efficient design and evaluation ofsuitable therapeutic solutions including individualized strategies foroptimizing drug usage, and screening of potential new medicamentcandidates.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the FLAP gene with an indication of the relativeposition of the biallelic markers of the present invention.

FIG. 2 show the results of an association study between the FLAPbiallelic markers and asthma with 290 asthmatic individuals and 280 USCaucasian controls. FIG. 2 is a graph demonstrating the associationbetween some of the biallelic markers of the invention and asthma withthe absolute value of the logarithm (base 10) of the p-value of thechi-square values for each marker shown on the y-axis and a roughestimate of the position of each marker with respect to the FLAP geneelements on the x-axis.

FIG. 3 is a table demonstrating the results of a haplotype associationanalysis between asthma and haplotypes which consist of biallelicmarkers of the invention. (297 cases vs 286 Caucasian US controls)

FIG. 4 is a table demonstrating the results of a haplotype frequencyanalysis including permutation testing with more than 1000 iterations.

BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE LISTING

SEQ ID NO: 1 contains a genomic sequence of FLAP comprising the 5′regulatory region (upstream untranscribed region), the exons andintrons, and the 3′ regulatory region (downstream untranscribed region).

SEQ ID NO: 2 contains a complete human FLAP cDNA with 5′ and 3′ UTRs.

SEQ ID NO: 3 contains the FLAP protein encoded by the cDNA of SEQ ID NO:2.

SEQ ID NOs: 4 and 5 contain either allele 1 or 2 of the biallelic markerA14 and its surrounding sequence.

SEQ ID NOs: 6 and 7 contain the sequence of amplification primers forthe biallelic marker A14.

SEQ ID NO: 8 contains the sequence of a microsequencing primer of thebiallelic marker A14.

SEQ ID NOs: 9 and 10 contain either allele 1 or 2 of the biallelicmarker A19 and its surrounding sequence.

SEQ ID NOs: 11 and 12 contain the sequence of amplification primers forthe biallelic marker A19.

SEQ ID NO: 13 contains the sequence of a microsequencing primer of thebiallelic marker A19.

SEQ ID NO: 14 contains a primer containing the additional PU 5′ sequencedescribed further in Example 2.

SEQ ID NO: 15 contains a primer containing the additional RP 5′ sequencedescribed further in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

5-LO is associated with FLAP for leukotriene synthesis. Indeed, itappears that regulation of the production of leukotrienes can beachieved either through the action of direct 5-LO inhibitors or indirectleukotriene biosynthesis inhibitors which bind to FLAP.

The present invention concerns the identification and characterizationof biallelic markers in a FLAP encoding gene, as well as theidentification of significant polymorphisms associated with diseasesinvolving the leukotriene pathway. Preferably, the polymorphisms areassociated with asthma.

The identified polymorphisms are used in the design of assays for thereliable detection of genetic susceptibility to diseases involving theleukotriene pathway. They can also be used in the design of drugscreening protocols to provide an accurate and efficient evaluation ofthe therapeutic and side-effect potential of new or already existingmedicaments.

I. Definitions

Before describing the invention in greater detail, the followingdefinitions are set forth to illustrate and define the meaning and scopeof the terms used to describe the invention herein.

The term “FLAP gene”, when used herein, encompasses genomic, mRNA andcDNA sequences encoding the FLAP protein. In the case of a genomicsequence, the FLAP gene also includes native regulatory regions whichcontrol the expression of the coding sequence of the FLAP gene.

As used interchangeably herein, the terms “oligonucleotides”, and“polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of morethan one nucleotide in either single chain or duplex form. The term“nucleotide” as used herein as an adjective to describe moleculescomprising RNA, DNA, or RNA/DNA hybrid sequences of any length insingle-stranded or duplex form. The term “nucleotide” is also usedherein as a noun to refer to individual nucleotides or varieties ofnucleotides, meaning a molecule, or individual unit in a larger nucleicacid molecule, comprising a purine or pyrimidine, a ribose ordeoxyribose sugar moiety, and a phosphate group, or phosphodiesterlinkage in the case of nucleotides within an oligonucleotide orpolynucleotide. Although the term “nucleotide” is also used herein toencompass “modified nucleotides” which comprise at least onemodifications (a) an alternative linking group, (b) an analogous form ofpurine, (c) an analogous form of pyrimidine, or (d) an analogous sugar,for examples of analogous linking groups, purine, pyrimidines, andsugars see for example PCT publication No WO 95/04064. However, thepolynucleotides of the invention are preferably comprised of greaterthan 50% conventional deoxyribose nucleotides, and most preferablygreater than 90% conventional deoxyribose nucleotides. Thepolynucleotide sequences of the invention may be prepared by any knownmethod, including synthetic, recombinant, ex vivo generation, or acombination thereof, as well as utilizing any purification methods knownin the art.

The term “purified” is used herein to describe a polynucleotide orpolynucleotide vector of the invention which has been separated fromother compounds including, but not limited to other nucleic acids,carbohydrates, lipids and proteins (such as the enzymes used in thesynthesis of the polynucleotide), or the separation of covalently closedpolynucleotides from linear polynucleotides. A polynucleotide issubstantially pure when at least about 50, preferably 60 to 75% of asample exhibits a single polynucleotide sequence and conformation(linear versus covalently closed). A substantially pure polynucleotidetypically comprises about 50, preferably 60 to 90% weight/weight of anucleic acid sample, more usually about 95%, and preferably is overabout 99% pure. Polynucleotide purity or homogeneity may be indicated bya number of means well known in the art, such as agarose orpolyacrylamide gel electrophoresis of a sample, followed by visualizinga single polynucleotide band upon staining the gel. For certain purposeshigher resolution can be provided by using HPLC or other means wellknown in the art.

As used herein, the term “isolated” requires that the material beremoved from its original environment (e.g., the natural environment ifit is naturally occurring). For example, a naturally-occurringpolynucleotide or polypeptide present in a living animal is notisolated, but the same polynucleotide or DNA or polypeptide, separatedfrom some or all of the coexisting materials in the natural system, isisolated. Such polynucleotide could be part of a vector and/or suchpolynucleotide or polypeptide could be part of a composition, and stillbe isolated in that the vector or composition is not part of its naturalenvironment.

The term “polypeptide” refers to a polymer of amino acids without regardto the length of the polymer; thus, peptides, oligopeptides, andproteins are included within the definition of polypeptide. This termalso does not specify or exclude post-expression modifications ofpolypeptides, for example, polypeptides which include the covalentattachment of glycosyl groups, acetyl groups, phosphate groups, lipidgroups and the like are expressly encompassed by the term polypeptide.Also included within the definition are polypeptides which contain oneor more analogs of an amino acid (including, for example, non-naturallyoccurring amino acids, amino acids which only occur naturally in anunrelated biological system, modified amino acids from mammalian systemsetc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring.

The term “purified” is used herein to describe a polypeptide of theinvention which has been separated from other compounds including, butnot limited to nucleic acids, lipids, carbohydrates and other proteins.A polypeptide is substantially pure when at least about 50%, preferably60 to 75% of a sample exhibits a single polypeptide sequence. Asubstantially pure polypeptide typically comprises about 50%, preferably60 to 90% weight/weight of a protein sample, more usually about 95%, andpreferably is over about 99% pure. Polypeptide purity or homogeneity isindicated by a number of means well known in the art, such as agarose orpolyacrylamide gel electrophoresis of a sample, followed by visualizinga single polypeptide band upon staining the gel. For certain purposeshigher resolution can be provided by using HPLC or other means wellknown in the art.

The term “recombinant polypeptide” is used herein to refer topolypeptides that have been artificially designed and which comprise atleast two polypeptide sequences that are not found as contiguouspolypeptide sequences in their initial natural environment, or to referto polypeptides which have been expressed from a recombinantpolynucleotide.

As used herein, the term “antibody” refers to a polypeptide or group ofpolypeptides which are comprised of at least one binding domain, wherean antibody binding domain is formed from the folding of variabledomains of an antibody molecule to form three-dimensional binding spaceswith an internal surface shape and charge distribution complementary tothe features of an antigenic determinant of an antigen, which allows animmunological reaction with the antigen. Antibodies include recombinantproteins comprising the binding domains, as wells as fragments,including Fab, Fab′, F(ab)₂, and F(ab′)₂ fragments.

As used herein, an “antigenic determinant” is the portion of an antigenmolecule, in this case a FLAP polypeptide, that determines thespecificity of the antigen-antibody reaction. An “epitope” refers to anantigenic determinant of a polypeptide. An epitope can comprise as fewas 3 amino acids in a spatial conformation which is unique to theepitope. Generally an epitope consists of at least 6 such amino acids,and more usually at least 8-10 such amino acids. Methods for determiningthe amino acids which make up an epitope include x-ray crystallography,2-dimensional nuclear magnetic resonance, and epitope mapping e.g. thePepscan method described by H. Mario Geysen et al. 1984; PCT PublicationNo WO 84/03564; and PCT Publication No WO 84/03506.

Throughout the present specification, the expression “nucleotidesequence” may be employed to designate indifferently a polynucleotide ora nucleic acid. More precisely, the expression “nucleotide sequence”encompasses the nucleic material itself and is thus not restricted tothe sequence information (i.e. the succession of letters chosen amongthe four base letters) that biochemically characterizes a specific DNAor RNA molecule.

The term “upstream” is used herein to refer to a location which istoward the 5′ end of the polynucleotide from a specific reference point.

The terms “base paired” and “Watson & Crick base paired” are usedinterchangeably herein to refer to nucleotides which can be hydrogenbonded to one another by virtue of their sequence identities in a mannerlike that found in double-helical DNA with thymine or uracil residueslinked to adenine residues by two hydrogen bonds and cytosine andguanine residues linked by three hydrogen bonds (See Stryer, L., 1995).

The terms “complementary” or “complement thereof” are used herein torefer to the sequences of polynucleotides which are capable of formingWatson & Crick base pairing with another specified polynucleotidethroughout the entirety of the complementary region. This term isapplied to pairs of polynucleotides based solely upon their sequencesand not any particular set of conditions under which the twopolynucleotides would actually bind.

The term “allele” is used herein to refer to variants of a nucleotidesequence. Diploid organisms may be homozygous or heterozygous for anallelic form.

A “promoter” refers to a DNA sequence recognized by the syntheticmachinery of the cell required to initiate the specific transcription ofa gene.

A sequence which is “operably linked” to a regulatory sequence such as apromoter means that said regulatory element is in the correct locationand orientation in relation to the nucleic acid to control RNApolymerase initiation and expression of the nucleic acid of interest. Asused herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the coding sequence. More precisely, twoDNA molecules (such as a polynucleotide containing a promoter region anda polynucleotide encoding a desired polypeptide or polynucleotide) aresaid to be “operably linked” if the nature of the linkage between thetwo polynucleotides does not (1) result in the introduction of aframe-shift mutation or (2) interfere with the ability of thepolynucleotide containing the promoter to direct the transcription ofthe coding polynucleotide.

The term “primer” denotes a specific oligonucleotide sequence which iscomplementary to a target nucleotide sequence and used to hybridize tothe target nucleotide sequence. A primer serves as an initiation pointfor nucleotide polymerization catalyzed by either DNA polymerase, RNApolymerase or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotideanalog segment, e.g., polynucleotide as defined hereinbelow) which canbe used to identify a specific polynucleotide sequence present insamples, said nucleic acid segment comprising a nucleotide sequencecomplementary of the specific polynucleotide sequence to be identified.

The term “heterozygosity rate” is used herein to refer to the incidenceof individuals in a population, which are heterozygous at a particularallele. In a biallelic system the heterozygosity rate is on averageequal to 2P_(a)(1−P_(a)), where P_(a) is the frequency of the leastcommon allele. In order to be useful in genetic studies a genetic markershould have an adequate level of heterozygosity to allow a reasonableprobability that a randomly selected person will be heterozygous.

The term “genotype” as used herein refers the identity of the allelespresent in an individual or a sample. In the context of the presentinvention a genotype preferably refers to the description of thebiallelic marker alleles present in an individual or a sample. The term“genotyping” a sample or an individual for a biallelic marker consistsof determining the specific allele or the specific nucleotide carried byan individual at a biallelic marker.

The term “mutation” as used herein refers to a difference in DNAsequence between or among different genomes or individuals which has afrequency below 1%.

The term “haplotype” refers to a combination of alleles present in anindividual or a sample. In the context of the present invention ahaplotype preferably refers to a combination of biallelic marker allelesfound in a given individual and which may be associated with aphenotype.

The term “polymorphism” as used herein refers to the occurrence of twoor more alternative genomic sequences or alleles between or amongdifferent genomes or individuals. “Polymorphic” refers to the conditionin which two or more variants of a specific genomic sequence can befound in a population. A “polymorphic site” is the locus at which thevariation occurs. A single nucleotide polymorphism is a single base pairchange. Typically a single nucleotide polymorphism is the replacement ofone nucleotide by another nucleotide at the polymorphic site. Deletionof a single nucleotide or insertion of a single nucleotide, also giverise to single nucleotide polymorphisms. In the context of the presentinvention “single nucleotide polymorphism” preferably refers to a singlenucleotide substitution. Typically, between different genomes or betweendifferent individuals, the polymorphic site may be occupied by twodifferent nucleotides.

“Biallelic markers” consist of a single base polymorphism. Eachbiallelic marker therefore corresponds to two forms of a polynucleotidesequence included in a gene, which, when compared with one another,present a nucleotide modification at one position. Usually, thenucleotide modification involves the substitution of one nucleotide foranother (for example C instead of T). Typically the frequency of theless common allele of the biallelic markers of the present invention hasbeen validated to be greater than 1%, preferably the frequency isgreater than 10%, more preferably the frequency is at least 20% (i.e.heterozygosity rate of at least 0.32), even more preferably thefrequency is at least 30% (i.e. heterozygosity rate of at least 0.42). Abiallelic marker wherein the frequency of the less common allele is 30%or more is termed a “high quality biallelic marker.”

As used herein the terminology “defining a biallelic marker” means thata sequence includes a polymorphic base from a biallelic marker. Thesequences defining a biallelic marker may be of any length consistentwith their intended use, provided that they contain a polymorphic basefrom a biallelic marker. The sequence has between 1 and 500 nucleotidesin length, preferably between 5, 10, 15, 20, 25, or 40 and 200nucleotides and more preferably between 30 and 50 nucleotides in length.Preferably, the sequences defining a biallelic marker include apolymorphic base selected from the group consisting of biallelic markersA1 to A28. In some embodiments the sequences defining a biallelic markercomprise one of the sequences selected from the group consisting of P1to P28. Likewise, the term “marker” or “biallelic marker” requires thatthe sequence is of sufficient length to practically (although notnecessarily unambiguously) identify the polymorphic allele, whichusually implies a length of at least 4, 5, 6, 10, 15, 20, 25, or 40nucleotides.

The invention also concerns FLAP-related biallelic markers. The term“FLAP-related biallelic marker” and “biallelic marker of the FLAP gene”are used interchangeably herein to relate to all biallelic markers inlinkage disequilibrium with the FLAP gene. The term FLAP-relatedbiallelic marker includes, but is not limited to, both the genic andnon-genic biallelic markers described in FIG. 1.

The term “non-genic” is used herein to describe FLAP-related biallelicmarkers, as well as polynucleotides and primers which occur outside thenucleotide positions shown in the human FLAP genomic sequence of SEQ IDNO: 1. The term “genic” is used herein to describe FLAP-relatedbiallelic markers as well as polynucleotides and primers which do occurin the nucleotide positions shown in the human FLAP genomic sequence ofSEQ ID NO: 1.

The location of nucleotides in a polynucleotide with respect to thecenter of the polynucleotide are described herein in the followingmanner. When a polynucleotide has an odd number of nucleotides, thenucleotide at an equal distance from the 3′ and 5′ ends of thepolynucleotide is considered to be “at the center” of thepolynucleotide, and any nucleotide immediately adjacent to thenucleotide at the center, or the nucleotide at the center itself isconsidered to be “within 1 nucleotide of the center.” With an odd numberof nucleotides in a polynucleotide any of the five nucleotides positionsin the middle of the polynucleotide would be considered to be within 2nucleotides of the center, and so on. When a polynucleotide has an evennumber of nucleotides, there would be a bond and not a nucleotide at thecenter of the polynucleotide. Thus, either of the two centralnucleotides would be considered to be “within 1 nucleotide of thecenter” and any of the four nucleotides in the middle of thepolynucleotide would be considered to be “within 2 nucleotides of thecenter”, and so on. For polymorphisms which involve the substitution,insertion or deletion of 1 or more nucleotides, the polymorphism, alleleor biallelic marker is “at the center” of a polynucleotide if thedifference between the distance from the substituted, inserted, ordeleted polynucleotides of the polymorphism and the 3′ end of thepolynucleotide, and the distance from the substituted, inserted, ordeleted polynucleotides of the polymorphism and the 5′ end of thepolynucleotide is zero or one nucleotide. If this difference is 0 to 3,then the polymorphism is considered to be “within 1 nucleotide of thecenter.” If the difference is 0 to 5, the polymorphism is considered tobe “within 2 nucleotides of the center.” If the difference is 0 to 7,the polymorphism is considered to be “within 3 nucleotides of thecenter,” and so on.

The terms “trait” and “phenotype” are used interchangeably herein andrefer to any visible, detectable or otherwise measurable property of anorganism such as symptoms of, or susceptibility to a disease forexample. Typically the terms “trait” or “phenotype” are used herein torefer to symptoms of, or susceptibility to a disease involving theleukotriene pathway; or to refer to an individual's response to an agentacting on the leukotriene pathway; or to refer to symptoms of, orsusceptibility to side-effects to an agent acting on the leukotrienepathway.

The term “disease involving the leukotriene pathway” refers to acondition linked to disturbances in expression, production or cellularresponse to leukotrienes. The diseases involving the leukotriene pathwayinclude, but are not limited to, such as angina, endotoxic shock,psoriasis, atopic eczema, rheumatoid arthritis, inflammatory boweldisease, osteoarthritis, tendinitis, bursitis, ulcerative colitis,allergic bronchoasthma, allergic rhinitis, allergic conjunctivitis,glomerulonephritis, migraine headaches, and more particularly asthma.

The terms “response to an agent acting on the leukotriene pathway” referto drug efficacy, including but not limited to ability to metabolize acompound, to the ability to convert a pro-drug to an active drug, and tothe pharmacokinetics (absorption, distribution, elimination) and thepharmacodynamics (receptor-related) of a drug in an individual. In thecontext of the present invention, a “positive response” to a medicamentcan be defined as comprising a reduction of the symptoms related to thedisease or condition to be treated. In the context of the presentinvention, a “negative response” to a medicament can be defined ascomprising either a lack of positive response to the medicament whichdoes not lead to a symptom reduction or to a side-effect observedfollowing administration of the medicament.

The terms “side-effects to an agent acting on the leukotriene pathway”refer to adverse effects of therapy resulting from extensions of theprincipal pharmacological action of the drug or to idiosyncratic adversereactions resulting from an interaction of the drug with unique hostfactors. The side-effects related to treatment with agents acting on theleukotriene pathway are preferably an acute exacerbation of aninflammatory disease such as asthma, infection and headache, and morepreferably an increase in liver transaminase levels.

The terms “agents acting on the leukotriene pathway” preferably refer toa drug or a compound which modulates the activity or concentration ofany enzyme or regulatory molecule involved in the leukotriene pathway ina cell or animal. Preferably these agents can be selected from thefollowing group: FLAP inhibitors such as BAYx 1005, MK-886, and MK-0591;5-Lipoxygenase inhibitors such as Zileuton, BAY-G576, RS-43,179,Wy-47,288, vitamin A, and BW A4C; Leukotriene LTD4 receptor antagonistssuch as zafirlukast, ICI-204,219, MK-571, MK-679, ONO-RS-411, SK&F104,353, and Wy-48,252; Leukotriene B4 receptor antagonists; LeukotrieneC4 synthase inhibitors; and, Leukotriene A4 hydrolase inhibitors.“Agents acting on the leukotriene pathway” further refers tonon-steroidal antiinflammatory drugs (NSAIDs), leukotriene receptorantagonists and leukotriene analogs. “Agents acting on the leukotrienepathway” also refers to compounds modulating the formation and action ofleukotrienes.

Some of the compounds cited above are described in U.S. Pat. Nos.4,873,259; 4,970,215; 5,310,744; 5,225,421; and 5,081,138; or in EP 0419 049, the disclosures of which are incorporated herein by reference.

The term “individual” as used herein refers to vertebrates, particularlymembers of the mammalian species and includes but is not limited todomestic animals, sports animals, laboratory animals, primates andhumans. Preferably, an individual is a human.

Variants and Fragments

Polynucleotides

The invention also relates to variants and fragments of thepolynucleotides described herein, particularly of a FLAP gene containingone or more biallelic markers according to the invention.

Variants of polynucleotides, as the term is used herein, arepolynucleotides that differ from a reference polynucleotide. A variantof a polynucleotide may be a naturally occurring variant such as anaturally occurring allelic variant, or it may be a variant that is notknown to occur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms. Generally, differencesare limited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical.

Changes in the nucleotide of a variant may be silent, which means thatthey do not alter the amino acids encoded by the polynucleotide.

However, nucleotide changes may also result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingor non-coding regions or both. Alterations in the coding regions mayproduce conservative or non-conservative amino acid substitutions,deletions or additions.

In the context of the present invention, particularly preferredembodiments are those in which the polynucleotides encode polypeptideswhich retain substantially the same biological function or activity asthe mature FLAP protein.

A polynucleotide fragment is a polynucleotide having a sequence thatentirely is the same as part but not all of a given nucleotide sequence,preferably the nucleotide sequence of a FLAP gene, and variants thereof.The fragment can be a portion of an exon or of an intron of a FLAP gene.It can also be a portion of the regulatory sequences of the FLAP gene.Preferably, such fragments comprise the polymorphic base of at least oneof the biallelic markers A1 to A28, the complement therefor, or abiallelic marker in linkage disequilibrium with one or more of thebiallelic markers A1 to A28.

Such fragments may be “free-standing”, i.e. not part of or fused toother polynucleotides, or they may be comprised within a single largerpolynucleotide of which they form a part or region. However, severalfragments may be comprised within a single larger polynucleotide.

As representative examples of polynucleotide fragments of the invention,there may be mentioned those which have from about 4, 6, 8, 15, 20, 25,40, 10 to 20, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or 100 to 200nucleotides in length. Preferred are those fragments having about 47nucleotides in length, such as those of P1 to P28, and containing atleast one of the biallelic markers of a FLAP gene which are describedherein. It will of course be understood that the polynucleotides P1 toP28 can be shorter or longer, although it is preferred that they atleast contain the polymorphic base of the biallelic marker which can belocated at one end of the fragment.

Polypeptides

The invention also relates to variants, fragments, analogs andderivatives of the polypeptides described herein, including mutated FLAPproteins.

The variant may be 1) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or2) one in which one or more of the amino acid residues includes asubstituent group, or 3) one in which the mutated FLAP is fused withanother compound, such as a compound to increase the half-life of thepolypeptide (for example, polyethylene glycol), or 4) one in which theadditional amino acids are fused to the mutated FLAP, such as a leaderor secretory sequence or a sequence which is employed for purificationof the mutated FLAP or a preprotein sequence. Such variants are deemedto be within the scope of those skilled in the art.

A polypeptide fragment is a polypeptide having a sequence that entirelyis the same as part but not all of a given polypeptide sequence,preferably a polypeptide encoded by a FLAP gene and variants thereof.Preferred fragments include those of the active region of the FLAPprotein that play a role in leukotriene biosynthesis and those regionspossessing antigenic properties and which can be used to raiseantibodies against the FLAP protein.

Such fragments may be “free-standing”, i.e. not part of or fused toother polypeptides, or they may be comprised within a single largerpolypeptide of which they form a part or region. However, severalfragments may be comprised within a single larger polypeptide.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 5, 6, 7, 8, 9 or 10to 15, 10 to 20, 15 to 40, or 30 to 55 amino acids long. Preferred arethose fragments containing at least one amino acid mutation in the FLAPprotein.

Stringent Hybridization Conditions

By way of example and not limitation, procedures using conditions ofhigh stringency are as follows: Prehybridization of filters containingDNA is carried out for 8 h to overnight at 65° C. in buffer composed of6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll,0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters arehybridized for 48 h at 65° C., the preferred hybridization temperature,in prehybridization mixture containing 100 μg/ml denatured salmon spermDNA and 5−20×10⁶ cpm of ³²P-labeled probe. Alternatively, thehybridization step can be performed at 65° C. in the presence of SSCbuffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate.Subsequently, filter washes can be done at 37° C. for 1 h in a solutioncontaining 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by awash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes canbe performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals.Following the wash steps, the hybridized probes are detectable byautoradiography. Other conditions of high stringency which may be usedare well known in the art and as cited in Sambrook et al., 1989; andAusubel et al., 1989, are incorporated herein in their entirety. Thesehybridization conditions are suitable for a nucleic acid molecule ofabout 20 nucleotides in length. There is no need to say that thehybridization conditions described above are to be adapted according tothe length of the desired nucleic acid, following techniques well knownto the one skilled in the art. The suitable hybridization conditions mayfor example be adapted according to the teachings disclosed in the bookof Hames and Higgins (1985) or in Sambrook et al. (1989).

Identity Between Nucleic Acids Or Polypeptides

The terms “percentage of sequence identity” and “percentage homology”are used interchangeably herein to refer to comparisons amongpolynucleotides and polypeptides, and are determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Homology is evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are by no means limited to, TBLASTN, BLASTP,FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al.,1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al.,1990; Altschul et al., 1993). In a particularly preferred embodiment,protein and nucleic acid sequence homologies are evaluated using theBasic Local Alignment Search Tool (“BLAST”) which is well known in theart (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990;Altschul et al., 1993; Altschul et al., 1997). In particular, fivespecific BLAST programs are used to perform the following task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against aprotein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotidesequence database;

(3) BLASTX compares the six-frame conceptual translation products of aquery nucleotide sequence (both strands) against a protein sequencedatabase;

(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and

(5) TBLASTX compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet etal., 1992; Henikoff and Henikoff, 1993). Less preferably, the PAM orPAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds.,1978). The BLAST programs evaluate the statistical significance of allhigh-scoring segment pairs identified, and preferably selects thosesegments which satisfy a user-specified threshold of significance, suchas a user-specified percent homology. Preferably, the statisticalsignificance of a high-scoring segment pair is evaluated using thestatistical significance formula of Karlin (see, e.g., Karlin andAltschul, 1990).

II. Genomic Sequences of FLAP

Although the FLAP gene is of high relevance to pharmaceutical research,we still have scant knowledge concerning the extent and nature ofsequence variation in this gene and its regulatory elements. The cDNAand part of the genomic sequence for human FLAP have been cloned andsequenced (Kennedy et al. 1991; Dixon et al, 1988). But, the completegenomic sequence of FLAP, including its regulatory elements, have notbeen described.

The present invention encompasses the genomic sequence of the FLAP geneof SEQ ID NO: 1 or a variant thereof or the complementary sequencethereto. This polynucleotide of nucleotide sequence of SEQ ID NO: 1, ora variant thereof or the complementary sequence thereto, may bepurified, isolated, or recombinant. The FLAP genomic sequences compriseexons and introns. The nucleic acids derived from the FLAP intronicpolynucleotides may be used as oligonucleotide primers or probes inorder to detect the presence of a copy of the FLAP gene in a testsample, or alternatively in order to amplify a target nucleotidesequence within the FLAP sequences.

The invention also encompasses a purified, isolated, or recombinantpolynucleotides comprising a nucleotide sequence having at least 70, 75,80, 85, 90, or 95% nucleotide identity with a nucleotide sequence of SEQID NO: 1 or a complementary sequence thereto or a fragment thereof. Thenucleotide differences as regards to the nucleotide sequence of SEQ IDNO: 1 may be generally randomly distributed throughout the entirenucleic acid. Nevertheless, preferred nucleic acids are those whereinthe nucleotide differences as regards to the nucleotide sequence of SEQID NO: 1 are predominantly located outside the coding sequencescontained in the exons. These nucleic acids, as well as their fragmentsand variants, may be used as oligonucleotide primers or probes in orderto detect the presence of a copy of the FLAP gene in a test sample, oralternatively in order to amplify a target nucleotide sequence withinthe FLAP sequences.

The FLAP genomic nucleic acid comprises 5 exons. Exon 1 starts at thenucleotide in position 7709 and ends at the nucleotide in position 7852of the nucleotide sequence of SEQ ID NO: 1; Exon 2 starts at thenucleotide in position 16236 and ends at the nucleotide in position16335 of the nucleotide sequence of SEQ ID NO: 1; Exon 3 starts at thenucleotide in position 24227 and ends at the nucleotide in position24297 of the nucleotide sequence of SEQ ID NO: 1; Exon 4 starts at thenucleotide in position 28133 and ends at the nucleotide in position28214 of the nucleotide sequence of SEQ ID NO: 1; Exon 5 starts at thenucleotide in position 36128 and ends at the nucleotide in position36605 of the nucleotide sequence of SEQ ID NO: 1. The invention alsodeals with purified, isolated, or recombinant nucleic acids comprising acombination of at least two exons of the FLAP gene, wherein thepolynucleotides are arranged within the nucleic acid, from the 5′-end tothe 3′-end of said nucleic acid, in the same order than in SEQ ID NO: 1.

The present invention also concerns a purified and/or isolated nucleicacid encoding a FLAP protein, preferably comprising at least one of thebiallelic polymorphisms described herewith, and more preferably a FLAPgene comprising the trait-causing mutation determined using thebelow-noted method. In some embodiments, the FLAP gene comprises one ormore of the sequences of P1 to P13, P15, and P17 to P28, or thecomplementary sequence thereto, or a fragment or a variant thereof.Preferred polynucleotides comprise at least one biallelic markerselected from the group consisting of A1 to A13, A15, A17 to A28, andthe complements thereof. The present invention also providespolynucleotides which, may be used as primers and probes in order toamplify fragments carrying biallelic markers or in order to detectbiallelic marker alleles.

Particularly preferred nucleic acids of the invention include isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID NO: 1 or the complementarysequence thereof, wherein said contiguous span comprises at least 1, 2,3, 5, or 10 of the following nucleotide positions of SEQ ID NO:1:1-7007, 8117-15994, 16550-24058, 24598-27872, 28413-35976, and36927-43069. Other preferred nucleic acids of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID NO: 1 or thecomplementary sequence thereof, wherein said contiguous span comprises aC at position 16348, of SEQ FD NO: 1. Further preferred nucleic acids ofthe invention include isolated, purified, or recombinant polynucleotidescomprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40,50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ IDNO: 1 or the complementary sequence thereof, wherein said contiguousspan comprises the following nucleotide positions of SEQ ID NO: 1:7612-7637, 24060-24061, 24067-24068, 27903-27905, and 28327-28329. Itshould be noted that nucleic acid fragments of any size and sequence mayalso be comprised by the polynucleotides described in this section.Additional preferred nucleic acids of the invention include isolated,purified, or recombinant polynucleotides comprising a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID NO: 1 or the complementarysequence thereof, wherein said contiguous span comprises a nucleotideselected from the group consisting of an A at position 7445, an A atposition 7870, a T at position 16288, an A at position 16383, a T atposition 24361, a G at position 28336, a T at position 28368, an A atposition 36183, and a G at position 36509 of SEQ ID NO: 1.

While this section is entitled “Genomic Sequences of FLAP,” it should benoted that nucleic acid fragments of any size and sequence may also becomprised by the polynucleotides described in this section, flanking thegenomic sequences of FLAP on either side or between two or more suchgenomic sequences.

Regulatory Regions of the FLAP Gene

The genomic sequence of the FLAP gene contains regulatory sequences bothin the non-coding 5′-flanking region and in the non-coding 3′-flankingregion that border the FLAP transcribed region containing the 5 exons ofthis gene. 5′-regulatory sequences of the FLAP gene comprise thepolynucleotide sequences located between the nucleotide in position 1and the nucleotide in position 7708 of the nucleotide sequence of SEQ IDNO: 1, more preferably between positions 1 and 7007 of SEQ ID NO: 1.3′-regulatory sequences of the FLAP gene comprise the polynucleotidesequences located between the nucleotide in position 36606 and thenucleotide in position 43069 of the nucleotide sequence of SEQ ID NO: 1.

Polynucleotides carrying the regulatory elements located both at the 5′end and at the 3′ end of the FLAP coding region may be advantageouslyused to control the transcriptional and translational activity of aheterologous polynucleotide of interest, said polynucleotide beingheterologous as regards to the FLAP regulatory region.

Thus, the present invention also concerns a purified, isolated, andrecombinant nucleic acid comprising a polynucleotide which is selectedfrom the group consisting of the polynucleotide sequences locatedbetween the nucleotide in position 1 and the nucleotide in position 7708of the nucleotide sequence of SEQ ID NO: 1, more preferably betweenpositions 1 and 7007 of SEQ ID NO: 1 and the polynucleotide sequenceslocated between the nucleotide in position 36606 and the nucleotide inposition 43069 of SEQ ID NO: 1; or a sequence complementary thereto or abiologically active fragment thereof.

The invention also pertains to a purified or isolated nucleic acidcomprising a polynucleotide having at least 95% nucleotide identity,advantageously 99% nucleotide identity, preferably 99.5% nucleotideidentity and most preferably 99.8% nucleotide identity with apolynucleotide selected from the group consisting of the polynucleotidesequences located between the nucleotide in position 1 and thenucleotide in position 7708 of the nucleotide sequence of SEQ ID NO: 1,more preferably between positions 1 and 7007 of SEQ ID NO: 1 and thepolynucleotide sequences located between the nucleotide in position36606 and the nucleotide in position 43069 of SEQ ID NO: 1 or a variantthereof or a biologically active fragment thereof.

Another object of the invention consists of purified, isolated orrecombinant nucleic acids comprising a polynucleotide that hybridizes,under the stringent hybridization conditions defined therein, with apolynucleotide selected from the group consisting of the polynucleotidesequences located between the nucleotide in position 1 and thenucleotide in position 7007 of SEQ ID NO: 1 and the polynucleotidesequences located between the nucleotide in position 36606 and thenucleotide in position 43069 of SEQ ID NO:, or a sequence complementarythereto or a variant thereof or a biologically active fragment thereof.

Furthermore, the present invention also concerns a purified, isolated,and recombinant nucleic acid comprising a polynucleotide which isselected from the group consisting of:

-   -   the polynucleotide sequences located between the nucleotide in        position 1 and the nucleotide in position 7708 of the nucleotide        sequence of SEQ ID NO: 1, more preferably between positions 1        and 7007 of SEQ ID NO: 1, and comprising a biallelic marker        selected from the group consisting of A1 to A11 and A25 to A28,        or a sequence complementary thereto or a biologically active        fragment thereof; and    -   the polynucleotide sequences located between the nucleotide in        position 36606 and the nucleotide in position 43069 of SEQ ID        NO: 1 and comprising a biallelic marker selected from the group        consisting of A22 to A24 and the complements thereof, or a        sequence complementary thereto or a biologically active fragment        thereof.

By a “biologically active” fragment of SEQ ID NO: 1 according to thepresent invention is intended a polynucleotide comprising oralternatively consisting of a fragment of said polynucleotide which isfunctional as a regulatory region for expressing a recombinantpolypeptide or a recombinant polynucleotide in a recombinant cell host.

For the purpose of the invention, a nucleic acid or polynucleotide is“functional” as a regulatory region for expressing a recombinantpolypeptide or a recombinant polynucleotide if said regulatorypolynucleotide contains nucleotide sequences which containtranscriptional and translational regulatory information, and suchsequences are “operably linked” to nucleotide sequences which encode thedesired polypeptide or the desired polynucleotide.

Preferred fragments of the 5′- or 3′-regulatory sequences have a lengthof about 1500 or 1000 nucleotides, preferably of about 500 nucleotides,more preferably about 400 nucleotides, even more preferably 300nucleotides and most preferably about 200 nucleotides.

The regulatory polynucleotides of the invention may be prepared from thepolynucleotide of SEQ ID NO: 1 by cleavage using suitable restrictionenzymes, as described for example in the book of Sambrook et al. (1989).The regulatory polynucleotides may also be prepared by digestion of thepolynucleotide of SEQ ID NO: 1 by an exonuclease enzyme, such as Bal31(Wabiko et al., 1986). These regulatory polynucleotides can also beprepared by nucleic acid chemical synthesis, as described elsewhere inthe specification.

The regulatory polynucleotides according to the invention may beadvantageously part of a recombinant expression vector that may be usedto express a coding sequence in a desired host cell or host organism.

A preferred 5′-regulatory polynucleotide of the invention includes the5′-untranslated region (5′-UTR) of the FLAP cDNA, or a biologicallyactive fragment or variant thereof.

A preferred 3′-regulatory polynucleotide of the invention includes the3′-untranslated region (3′-UTR) of the FLAP cDNA, or a biologicallyactive fragment or variant thereof.

A further object of the invention consists of an isolated, purified orrecombined polynucleotide comprising:

a) a nucleic acid comprising a regulatory nucleotide sequence selectedfrom the group consisting of:

(i) a polynucleotide beginning at position 1 and ending at position 7708of SEQ ID NO: 1, more preferably beginning at position 1 and ending atposition 7007 of SEQ ID NO: 1, or a sequence complementary thereto;

(ii) a polynucleotide having at least 95% of nucleotide identity withthe nucleotide sequence beginning at position 1 and ending at position7708 of SEQ ID NO: 1, more preferably beginning at position 1 and endingat position 7007 of SEQ ID NO: 1, or a sequence complementary thereto;

(iii) a polynucleotide that hybridizes under stringent hybridizationconditions with the nucleotide sequence beginning at position 1 andending at position 7007 of SEQ ID NO: 1, or a sequence complementarythereto;

(iv) a biologically active fragment or variant of the polynucleotides in(i), (ii) and (iii);

b) a polynucleotide encoding a desired polypeptide or a nucleic acid ofinterest, operably linked to the nucleic acid defined in (a) above;

c) Optionally, a nucleic acid comprising a 3′-regulatory polynucleotide,preferably a 3′-regulatory polynucleotide of the FLAP gene.

In a specific embodiment of the nucleic acid defined above, said nucleicacid includes the 5′-untranslated region (5′-UTR) of the FLAP cDNA, or abiologically active fragment or variant thereof.

In a second specific embodiment of the nucleic acid defined above, saidnucleic acid includes the 3′-untranslated region (3′-UTR) of the FLAPcDNA, or a biologically active fragment or variant thereof.

The regulatory sequences may comprise a biallelic marker selected fromthe group consisting of A1 to A11 and A22 to A28, and the complementsthereof.

The polypeptide encoded by the nucleic acid described above may be ofvarious nature or origin, encompassing proteins of prokaryotic oreukaryotic origin. Among the polypeptides expressed under the control ofa FLAP regulatory region, there may be cited bacterial, fungal or viralantigens. Also encompassed are eukaryotic proteins such as intracellularproteins, for example “house keeping” proteins, membrane-bound proteins,for example receptors, and secreted proteins, for example cytokines. Ina specific embodiment, the desired polypeptide may be the FLAP protein,especially the protein of the amino acid sequence of SEQ ID NO: 3.

The desired nucleic acids encoded by the above described polynucleotide,usually a RNA molecule, may be complementary to a desired codingpolynucleotide, for example to the FLAP coding sequence, and thus usefulas an antisense polynucleotide.

Such a polynucleotide may be included in a recombinant expression vectorin order to express the desired polypeptide or the desired nucleic acidin host cell or in a host organism.

III. FLAP cDNA Sequences

The present invention provides a FLAP cDNA of SEQ ID NO: 2. The cDNA ofSEQ ID NO: 2 also includes a 5′-UTR region and a 3′-UTR region. The5′-UTR region starts at the nucleotide at position 1 and ends at thenucleotide in position 74 of SEQ ID NO: 2. The 3′-UTR region starts atthe nucleotide at position 561 and ends at the nucleotide at position875 of SEQ ID NO: 2. The polyadenylation site starts at the nucleotideat position 851 and ends at the nucleotide in position 856 of SEQ ID NO:2.

Consequently, the invention concerns a purified, isolated, andrecombinant nucleic acids comprising a nucleotide sequence of the 5′UTRand the 3′UTR of the FLAP cDNA, a sequence complementary thereto, or anallelic variant thereof.

Another object of the invention is a purified, isolated, or recombinantnucleic acid comprising the nucleotide sequence of SEQ ID NO: 2,complementary sequences thereto or a variant or fragment thereof.Moreover, preferred polynucleotides of the invention include purified,isolated, or recombinant FLAP cDNAs consisting of, consistingessentially of, or comprising the sequence of SEQ ID NO: 2. A particularpreferred embodiment of the invention includes isolated, purified, orrecombinant polynucleotides comprising a contiguous span of at least 12,15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or1000 nucleotides of SEQ ID NO: 2 or a complementary sequence thereto,wherein said contiguous span comprises a T at position 197 (A13), an Aat position 453 (A20), or a G at position 779 (A21) of SEQ ID NO: 2.

Most biallelic polymorphisms represent silent nucleotide substitutionsbut biallelic marker A20 is associated with amino acid changes fromvaline to isoleucine in position 127 in the corresponding FLAPpolypeptide.

The polynucleotide disclosed above that contains the coding sequence ofthe FLAP gene of the invention may be expressed in a desired host cellor a desired host organism, when this polynucleotide is placed under thecontrol of suitable expression signals. The expression signals may beeither the expression signals contained in the regulatory regions in theFLAP gene of the invention or may be exogenous regulatory nucleicsequences. Such a polynucleotide, when placed under the suitableexpression signals, may also be inserted in a vector for its expression.

While this section is entitled “FLAP cDNA Sequences,” it should be notedthat nucleic acid fragments of any size and sequence may also becomprised by the polynucleotides described in this section, flanking thegenomic sequences of FLAP on either side or between two or more suchgenomic sequences.

Coding Regions

The FLAP open reading frame is contained in the corresponding mRNA ofSEQ ID NO: 2. More precisely, the effective FLAP coding sequence (CDS)spans from the nucleotide in position 75 (first nucleotide of the ATGcodon) to the nucleotide in position 560 (end nucleotide of the TGAcodon) of the polynucleotide sequence of SEQ ID NO: 2. The presentinvention also embodies isolated, purified, and recombinantpolynucleotides which encode a polypeptide comprising a contiguous spanof at least 6 amino acids, preferably at least 8 or 10 amino acids, morepreferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids ofSEQ ID NO: 3, wherein said contiguous span includes a isoleucine residueat amino acid position 127 in SEQ ID NO: 3.

The above disclosed polynucleotide that contains the coding sequence ofthe FLAP gene may be expressed in a desired host cell or a desired hostorganism, when this polynucleotide is placed under the control ofsuitable expression signals. The expression signals may be either theexpression signals contained in the regulatory regions in the FLAP geneof the invention or in contrast the signals may be exogenous regulatorynucleic sequences. Such a polynucleotide, when placed under the suitableexpression signals, may also be inserted in a vector for its expressionand/or amplification.

IV. Polynucleotide Constructs

The terms “polynucleotide construct” and “recombinant polynucleotide”are used interchangeably herein to refer to linear or circular, purifiedor isolated polynucleotides that have been artificially designed andwhich comprise at least two nucleotide sequences that are not found ascontiguous nucleotide sequences in their initial natural environment.

DNA Construct that Enables Directing Temporal and Spatial Flap GeneExpression in Recombinant Cell Hosts and in Transgenic Animals.

In order to study the physiological and phenotypic consequences of alack of synthesis of the FLAP protein, both at the cell level and at themulti cellular organism level, the invention also encompasses DNAconstructs and recombinant vectors enabling a conditional expression ofa specific allele of the FLAP genomic sequence or cDNA and also of acopy of this genomic sequence or cDNA harboring substitutions,deletions, or additions of one or more bases as regards to the FLAPnucleotide sequence of SEQ ID NOs: 1 and 2, or a fragment thereof, thesebase substitutions, deletions or additions being located either in anexon, an intron or a regulatory sequence, but preferably in the5′-regulatory sequence or in an exon of the FLAP genomic sequence orwithin the FLAP cDNA of SEQ ID NO: 2. In a preferred embodiment, theFLAP sequence comprises a biallelic marker of the present invention,preferably one of the biallelic markers A1 to A28.

The present invention embodies recombinant vectors comprising any one ofthe polynucleotides described in the present invention.

A first preferred DNA construct is based on the tetracycline resistanceoperon tet from E. coli transposon Tn110 for controlling the FLAP geneexpression, such as described by Gossen et al. (1992, 1995) and Furth etal. (1994). Such a DNA construct contains seven tet operator sequencesfrom Tn10 (tetop) that are fused to either a minimal promoter or a5′-regulatory sequence of the FLAP gene, said minimal promoter or saidFLAP regulatory sequence being operably linked to a polynucleotide ofinterest that codes either for a sense or an antisense oligonucleotideor for a polypeptide, including a FLAP polypeptide or a peptide fragmentthereof. This DNA construct is functional as a conditional expressionsystem for the nucleotide sequence of interest when the same cell alsocomprises a nucleotide sequence coding for either the wild type (tTA) orthe mutant (rTA) repressor fused to the activating domain of viralprotein VP16 of herpes simplex virus, placed under the control of apromoter, such as the HCMVIE1 enhancer/promoter or the MMTV-LTR. Indeed,a preferred DNA construct of the invention comprise both thepolynucleotide containing the tet operator sequences and thepolynucleotide containing a sequence coding for the tTA or the rTArepressor.

In a specific embodiment, the conditional expression DNA constructcontains the sequence encoding the mutant tetracycline repressor rTA,the expression of the polynucleotide of interest is silent in theabsence of tetracycline and induced in its presence.

DNA Constructs Allowing Homologous Recombination: Replacement Vectors

A second preferred DNA construct will comprise, from 5′-end to 3′-end:(a) a first nucleotide sequence that is comprised in the FLAP genomicsequence; (b) a nucleotide sequence comprising a positive selectionmarker, such as the marker for neomycin resistance (neo); and (c) asecond nucleotide sequence that is comprised in the FLAP genomicsequence, and is located on the genome downstream the first FLAPnucleotide sequence (a).

In a preferred embodiment, this DNA construct also comprises a negativeselection marker located upstream the nucleotide sequence (a) ordownstream the nucleotide sequence (c). Preferably, the negativeselection marker consists of the thymidine kinase (tk) gene (Thomas etal., 1986), the hygromycin beta gene (Te Riele et al., 1990), the hprtgene (Van der Lugt et al., 1991; Reid et al., 1990) or the Diphtheriatoxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al. 1990).Preferably, the positive selection marker is located within a FLAP exonsequence so as to interrupt the sequence encoding a FLAP protein. Thesereplacement vectors are described, for example, by Thomas et al. (1986;1987), Mansour et al. (1988) and Koller et al. (1992).

The first and second nucleotide sequences (a) and (c) may beindifferently located within a FLAP regulatory sequence, an intronicsequence, an exon sequence or a sequence containing both regulatoryand/or intronic and/or exon sequences. The size of the nucleotidesequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.

DNA Constructs Allowing Homologous Recombination: Cre-LoxP System.

These new DNA constructs make use of the site specific recombinationsystem of the PI phage. The PI phage possesses a recombinase called Crewhich interacts specifically with a 34 base pairs loxP site. The loxPsite is composed of two palindromic sequences of 13 bp separated by a 8bp conserved sequence (Hoess et al., 1986). The recombination by the Creenzyme between two loxP sites having an identical orientation leads tothe deletion of the DNA fragment.

The Cre-loxP system used in combination with a homologous recombinationtechnique has been first described by Gu et al. (1993, 1994). Briefly, anucleotide sequence of interest to be inserted in a targeted location ofthe genome harbors at least two loxP sites in the same orientation andlocated at the respective ends of a nucleotide sequence to be excisedfrom the recombinant genome. The excision event requires the presence ofthe recombinase (Cre) enzyme within the nucleus of the recombinant cellhost. The recombinase enzyme may be brought at the desired time eitherby (a) incubating the recombinant cell hosts in a culture mediumcontaining this enzyme, by injecting the Cre enzyme directly into thedesired cell, such as described by Araki et al. (1995), or bylipofection of the enzyme into the cells, such as described by Bauboniset al. (1993); (b) transfecting the cell host with a vector comprisingthe Cre coding sequence operably linked to a promoter functional in therecombinant cell host, which promoter being optionally inducible, saidvector being introduced in the recombinant cell host, such as describedby Gu et al. (1993) and Sauer et al. (1988); (c) introducing in thegenome of the cell host a polynucleotide comprising the Cre codingsequence operably linked to a promoter functional in the recombinantcell host, which promoter is optionally inducible, and saidpolynucleotide being inserted in the genome of the cell host either by arandom insertion event or an homologous recombination event, such asdescribed by Gu et al. (1994).

In a specific embodiment, the vector containing the sequence to beinserted in the FLAP gene by homologous recombination is constructed insuch a way that selectable markers are flanked by loxP sites of the sameorientation, it is possible, by treatment by the Cre enzyme, toeliminate the selectable markers while leaving the FLAP sequences ofinterest that have been inserted by an homologous recombination event.Again, two selectable markers are needed: a positive selection marker toselect for the recombination event and a negative selection marker toselect for the homologous recombination event. Vectors and methods usingthe Cre-loxP system are described by Zou et al. (1994).

Thus, a third preferred DNA construct of the invention comprises, from5′-end to 3′-end: (a) a first nucleotide sequence that is comprised inthe FLAP genomic sequence; (b) a nucleotide sequence comprising apolynucleotide encoding a positive selection marker, said nucleotidesequence comprising additionally two sequences defining a siterecognized by a recombinase, such as a loxP site, the two sites beingplaced in the same orientation; and (c) a second nucleotide sequencethat is comprised in the FLAP genomic sequence, and is located on thegenome downstream of the first FLAP nucleotide sequence (a).

The sequences defining a site recognized by a recombinase, such as aloxP site, are preferably located within the nucleotide sequence (b) atsuitable locations bordering the nucleotide sequence for which theconditional excision is sought. In one specific embodiment, two loxPsites are located at each side of the positive selection markersequence, in order to allow its excision at a desired time after theoccurrence of the homologous recombination event.

In a preferred embodiment of a method using the third DNA constructdescribed above, the excision of the polynucleotide fragment bordered bythe two sites recognized by a recombinase, preferably two loxP sites, isperformed at a desired time, due to the presence within the genome ofthe recombinant host cell of a sequence encoding the Cre enzyme operablylinked to a promoter sequence, preferably an inducible promoter, morepreferably a tissue-specific promoter sequence and most preferably apromoter sequence which is both inducible and tissue-specific, such asdescribed by Gu et al. (1994).

The presence of the Cre enzyme within the genome of the recombinant cellhost may be the result of the breeding of two transgenic animals, thefirst transgenic animal bearing the FLAP-derived sequence of interestcontaining the loxP sites as described above and the second transgenicanimal bearing the Cre coding sequence operably linked to a suitablepromoter sequence, such as described by Gu et al. (1994).

Spatio-temporal control of the Cre enzyme expression may also beachieved with an adenovirus based vector that contains the Cre gene thusallowing infection of cells, or in vivo infection of organs, fordelivery of the Cre enzyme, such as described by Anton and Graham (1995)and Kanegae et al. (1995).

The DNA constructs described above may be used to introduce a desirednucleotide sequence of the invention, preferably a FLAP genomic sequenceor a FLAP cDNA sequence, and most preferably an altered copy of a FLAPgenomic or cDNA sequence, within a predetermined location of thetargeted genome, leading either to the generation of an altered copy ofa targeted gene (knock-out homologous recombination) or to thereplacement of a copy of the targeted gene by another copy sufficientlyhomologous to allow an homologous recombination event to occur (knock-inhomologous recombination). In a specific embodiment, the DNA constructsdescribed above may be used to introduce a FLAP genomic sequence or aFLAP cDNA sequence comprising at least one biallelic marker of thepresent invention, preferably at least one biallelic marker selectedfrom the group consisting of A1 to A28 and the complements thereof, morepreferably at least one biallelic marker selected from the groupconsisting of A1 to A13, A15, and A17 to A28 and the complementsthereof.

Nuclear Antisense DNA Constructs

Other compositions containing a vector of the invention comprise anoligonucleotide fragment of the nucleic sequence SEQ ID NO: 2 comprisinga biallelic marker of the invention, preferably a fragment including thestart codon of the FLAP gene, as an antisense tool that inhibits theexpression of the corresponding FLAP gene. Preferred methods usingantisense polynucleotide according to the present invention are theprocedures described by Sczakiel et al. (1995) or those described in PCTApplication No WO 95/24223.

Preferably, the antisense tools are chosen among the polynucleotides(15-200 bp long) that are complementary to the 5′ end of the FLAP mRNA.In one embodiment, a combination of different antisense polynucleotidescomplementary to different parts of the desired targeted gene are used.

Preferred antisense polynucleotides according to the present inventionare complementary to a sequence of the mRNAs of FLAP that containseither the translation initiation codon ATG or a splicing site. Furtherpreferred antisense polynucleotides according to the invention arecomplementary of the splicing site of the FLAP mRNA.

Preferably, the antisense polynucleotides of the invention have a 3′polyadenylation signal that has been replaced with a self-cleavingribozyme sequence, such that RNA polymerase II transcripts are producedwithout poly(A) at their 3′ ends, these antisense polynucleotides beingincapable of export from the nucleus, such as described by Liu et al.(1994). In a preferred embodiment, these FLAP antisense polynucleotidesalso comprise, within the ribozyme cassette, a histone stem-loopstructure to stabilize cleaved transcripts against 3′-5′ exonucleolyticdegradation, such as the structure described by Eckner et al. (1991).

V. Biallelic Markers of the FLAP Gene

The invention also concerns FLAP-related biallelic markers, preferably abiallelic marker associated with a disease involving the leukotrienepathway, most preferably asthma. The term FLAP-related biallelic markerincludes the biallelic markers designated A1 to A28. The invention alsoconcerns sets of these biallelic markers.

28 biallelic markers have been identified in the genomic sequence ofFLAP. These biallelic markers are disclosed in Table 2 of Example 3.Their location on the FLAP genomic sequence and cDNA is indicated inTable 2 and also as a single base polymorphism in the features of SEQ IDNO: 1. Table 2 also discloses the position in SEQ ID NO: 1 ofpolynucleotides of 47 nucleotides in length, designated P1 to P28, whichcomprise a biallelic marker of the FLAP gene and define said biallelicmarker. The pairs of primers allowing the amplification of a nucleicacid containing the polymorphic base of one FLAP biallelic marker arelisted in Table 1 of Example 2. Three biallelic markers, namely A13, A20and A21, are located in exonic regions. Two of them do not modify theamino acid sequence of the FLAP protein. However, the biallelic markerA20 changes a valine into a isoleucine in the FLAP protein.

The invention also relates to a purified and/or isolated nucleotidesequence comprising a polymorphic base of a biallelic marker located inthe sequence of the FLAP gene, preferably of a biallelic marker selectedfrom the group consisting of A1 to A28, preferably from the groupconsisting of A1 to A13, A15, and A17 to A28, and the complementsthereof, optionally, said biallelic marker is selected from the groupconsisting of A1 to A10 and A22 to A28; optionally, said biallelicmarker is selected from the group consisting of A11 to A13, A15, A17 toA21; optionally, said biallelic marker is either A14 or A16. Thesequence has between 8 and 1000 nucleotides in length, and preferablycomprises at least 8, 10, 12, 15, 18, 20, 25, 35, 40, 50, 60, 70, 80,100, 250, 500 or 1000 contiguous nucleotides of a nucleotide sequenceselected from the group consisting of SEQ ID NOs: 1 and 2 or a variantthereof or a complementary sequence thereto. These nucleotide sequencescomprise the polymorphic base of either allele 1 or allele 2 of theconsidered biallelic marker. Optionally, said biallelic marker may bewithin 6, 5, 4, 3, 2, or 1 nucleotides of the center of saidpolynucleotide or at the center of said polynucleotide. Optionally, the3′ end of said contiguous span may be present at the 3′ end of saidpolynucleotide. Optionally, the biallelic marker may be present at the3′ end of said polynucleotide. Optionally, the 3′ end of saidpolynucleotide may be located within or at least 2, 4, 6, 8, 10, 12, 15,18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of abiallelic marker of the FLAP gene in said sequence. Optionally, the 3′end of said polynucleotide may be located 1 nucleotide upstream of abiallelic marker of the FLAP gene in said sequence. Optionally, saidpolynucleotide may further comprise a label. Optionally, saidpolynucleotide can be attached to solid support. In a furtherembodiment, the polynucleotides defined above can be used alone or inany combination.

The invention further concerns a nucleic acid encoding the FLAP protein,wherein said nucleic acid comprises a polymorphic base of a biallelicmarker selected from the group consisting of A1 to A28 and thecomplements thereof, preferably from the group consisting of A1 to A13,A15, and A17 to A28 and the complements thereof.

The invention also relates to a nucleotide sequence, preferably apurified and/or isolated nucleotide sequence comprising a sequencedefining a biallelic marker of the FLAP gene, a fragment or variantthereof or a sequence complementary thereto, said fragment comprisingthe polymorphic base. Preferably, the sequences defining a biallelicmarker include the polymorphic base of one of the polynucleotides P1 toP13, P15 and P17 to P28 or the complements thereof. In some embodiments,the sequences defining a biallelic marker comprise a nucleotide sequenceselected from the group consisting of P1 to P13, P15 and P17 to P28, andthe complementary sequence thereto or a fragment thereof, said fragmentcomprising the polymorphic base.

The invention also concerns a set of the purified and/or isolatednucleotide sequences defined above. More particularly, the set ofpurified and/or isolated nucleotide sequences comprises a group ofsequences defining a combination of biallelic markers of the FLAP gene.Preferably, the combination of alleles of biallelic markers isassociated with asthma.

In a preferred embodiment, the invention relates to a set of purifiedand/or isolated nucleotide sequences, each sequence comprising asequence defining a biallelic marker of the FLAP gene, wherein the setis characterized in that between about 30 and 100%, preferably betweenabout 40 and 60%, more preferably between 50 and 60%, of the sequencesdefining a biallelic marker are selected from the group consisting of P1to P28, preferably of P1 to P13, P15 and P17 to P28, or a fragment orvariant thereof or the complementary sequence thereto, said fragmentcomprising the polymorphic base.

More particularly, the invention concerns a set of purified and/orisolated nucleotide sequences, each sequence comprising a sequencedefining a different biallelic marker of the FLAP gene, said biallelicmarker being either included in a nucleotide sequence selected from thegroup consisting of P1 to P28 and the complementary sequence thereto,preferably of P1 to P13, P15 and P17 to P28 and the complementarysequence thereto, or a biallelic marker, preferably one located in thesequence of the FLAP gene, biallelic markers A1 to A28, or markers inlinkage disequilibrium with one of the markers of the set definedherewith.

The invention also relates to a set of at least two, preferably four,five, six, seven, eight or more nucleotide sequences selected from thegroup consisting of P1 to P28, preferably of P1 to P13, P15 and P17 toP28, and the complementary sequence thereto, or a fragment or variantthereof, said fragment comprising the polymorphic base. Preferably, thisset comprises at least one nucleotide sequence defining a biallelicmarker for each linkage disequilibrium region of the FLAP gene.

The invention further concerns a nucleotide sequence selected from thegroup consisting of P1 to P13, P15 and P17 to P28, or a complementarysequence thereto or a fragment or a variant thereof, said fragmentcomprising the polymorphic base.

In a further embodiment, the sequences comprising a polymorphic base ofone of the biallelic markers listed in Table 2 are selected from thegroup consisting of the nucleotide sequences that have a contiguous spanof, that consist of, that are comprised in, or that comprises apolynucleotide selected from the group consisting of the nucleic acidsof the sequences set forth as Nos. 10-517, 10-518, 10-253, 10-499,10-500, 10-522, 10-503, 10-504, 10-204, 10-32, 10-33, 10-34, 10-35,10-36, 10-498, 12-628, and 12-629 (listed in Table 1) or a variantthereof or a complementary sequence thereto.

VI. Oligonucleotide Probes and Primers

Polynucleotides derived from the FLAP gene are useful in order to detectthe presence of at least a copy of a nucleotide sequence of SEQ ID NO: 1or 2, or a fragment or a variant thereof in a test sample.

Particularly preferred probes and primers comprise a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID NO: 1 or the complementarysequence thereof, wherein said contiguous span comprises at least 1, 2,3, 5, or 10 of the following nucleotide positions of SEQ ID NO:1:1-7007, 8117-15994, 16550-24058, 24598-27872, 28413-35976, and36927-43069. Other preferred nucleic acids of the invention includeisolated, purified, or recombinant polynucleotides comprising acontiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70,80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID NO: 1 or thecomplementary sequence thereof, wherein said contiguous span comprises aC at position 16348, of SEQ ID NO: 1. Further preferred nucleic acids ofthe invention include isolated, purified, or recombinant polynucleotidescomprising a contiguous span of at least 26, 30, 35, 40, 50, 60, 70, 80,90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID NO: 1 or thecomplementary sequence thereof, wherein said contiguous span comprisesof the following nucleotide positions of SEQ ID NO: 1: 7612-7637,24060-24061, 24067-24068, 27903-27905, and 28327-28329. Additionalpreferred probes and primers comprise a contiguous span of at least 12,15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or1000 nucleotides of SEQ ID NO: 1 or the complementary sequence thereof,wherein said contiguous span comprises a nucleotide selected from thegroup consisting of an A at position 7445, an A at position 7870, a T atposition 16288, an A at position 16383, a T at position 24361, a G atposition 28336, a T at position 28368, an A at position 36183, and a Gat position 36509 of SEQ ID NO: 1.

Thus, the invention also relates to nucleic acid probes or primerscharacterized in that they hybridize specifically, under the stringenthybridization conditions defined above, with a nucleic acid selectedfrom the group consisting of the nucleotide sequences 1-7007,8117-15994, 16550-24058, 24598-27872, 28413-35976, and 36927-43069 ofSEQ ID NO: 1 or a variant thereof or a sequence complementary thereto.

Particularly preferred probes and primers comprise a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 500, or 1000 nucleotides of SEQ ID NO: 2 or a complementarysequence thereto, wherein said contiguous span comprises a T at position197 (A13), an A at position 453 (A20), or a G at position 779 (A21) ofSEQ ID NO: 2.

The present invention also concerns oligonucleotides and groups ofoligonucleotides for the detection of alleles associated with a modifiedleukotriene metabolism, preferably alleles associated with a FLAP genepolymorphism, and more preferably alleles of a FLAP gene associated witha disease involving the leukotriene pathway, for example asthma. Theseoligonucleotides are characterized in that they can hybridize with aFLAP gene, preferably with a polymorphic FLAP gene and more preferablywith a region of a FLAP gene comprising the polymorphic site of whichspecific alleles are associated with a disease involving the leukotrienepathway such as asthma. The oligonucleotides are useful either asprimers for use in various processes such as DNA amplification andmicrosequencing or as probes for DNA recognition in hybridizationanalyses. In some embodiments, the oligonucleotides contain thepolymorphic base of a sequence selected from the group consisting of P1to P28 and the complementary sequence thereto, more preferably from thegroup consisting of P1 to P13, P15, P17 to P28 and the complementarysequence thereto. In other embodiments, the oligonucleotides have a 3′terminus immediately adjacent to a polymorphic base in the FLAP gene,such as a polymorphic base in one of P1 to P28 and the complementarysequence thereto, optionally of P1 to P13, P15, and P17 to P28 and thecomplementary sequence thereto. In other embodiments, theoligonucleotide is capable of discriminating between different allelesof a biallelic marker in the FLAP gene, said biallelic marker beingselected from the group consisting of A1 to A28 and the complementsthereof, optionally of A1 to A13, A15, and A17 to A28 and thecomplements thereof. For example, the oligonucleotide may be capable ofspecifically hybridizing to one allele of a biallelic marker, includingone of the biallelic markers A1 to A28 and the complements thereof,optionally of A1 to A13, A15, and A17 to A28 and the complementsthereof. In another embodiment, the oligonucleotides comprise one of thesequences of B1 to B17, C1 to C17, D1 to D28, E1 to E28, and P1 to P28,and the complementary sequence thereto. Optionally, the oligonucleotidescomprise one of the sequences of B1 to B17, C1 to C17, D1 to D13, D15,D17 to D28, E1 to E13, E15, E17 to E28, P1 to P13, P15, and P17 to P28,and the complementary sequence thereto.

In one embodiment the invention encompasses isolated, purified, andrecombinant polynucleotides consisting of, or consisting essentially ofa contiguous span of 8 to 50 nucleotides of SEQ ID NO: 1 or 2 and thecomplement thereof, wherein said span includes a FLAP-related biallelicmarker in said sequence; optionally, wherein said FLAP-related biallelicmarker is selected from the group consisting of A1 to A28, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, wherein said FLAP-relatedbiallelic marker is selected from the group consisting of A1 to A13,A15, A17 to A28, and the complements thereof; or optionally thebiallelic markers in linkage disequilibrium therewith; optionally,wherein said FLAP-related biallelic marker is selected from the groupconsisting of A1 to A10 and A22 to A28, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith;optionally, wherein said FLAP-related biallelic marker is selected fromthe group consisting of A11 to A13, A15, A17 to A21, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, wherein said FLAP-related biallelic marker isselected from the group consisting of A14 and A16, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, wherein said contiguous span is 18 to 47nucleotides in length and said biallelic marker is within 6, 5, 4, 3, 2,or 1 nucleotides of the center of the polynucleotide and preferablywithin 4 nucleotides of the center of said polynucleotide; optionally,wherein said polynucleotide consists of said contiguous span and saidcontiguous span is 25 nucleotides in length and said biallelic marker isat the center of said polynucleotide; optionally, wherein the 3′ end ofsaid contiguous span is present at the 3′ end of said polynucleotide;and optionally, wherein the 3′ end of said contiguous span is located atthe 3′ end of said polynucleotide and said biallelic marker is presentat the 3′ end of said polynucleotide.

In another embodiment the invention encompasses isolated, purified andrecombinant polynucleotides consisting of, or consisting essentially ofa contiguous span of 8 to 50 nucleotides of SEQ ID NO: 1 or 2 or thecomplement thereof, wherein the 3′ end of said contiguous span islocated at the 3′ end of said polynucleotide. In one embodiment, the 3′end of said polynucleotide is located within or at least 2, 4, 6, 8, 10,12, 15, 18, 20, 25, 50, 100, 250, 500, or 1000 nucleotides upstream of abiallelic marker of FLAP in said sequence or at any other location whichis appropriate for their intended use in sequencing, amplification orthe location of novel sequences or markers. In a particular embodiment,the 3′ end of said polynucleotide is located within 20 nucleotidesupstream of a FLAP-related biallelic marker in said sequence;optionally, wherein said FLAP-related biallelic marker is selected fromthe group consisting of A1 to A28, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith;optionally, wherein said FLAP-related biallelic marker is selected fromthe group consisting of A1 to A13, A15, A17 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, wherein said FLAP-related biallelic marker isselected from the group consisting of A1 to A10 and A22 to A28, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, wherein said FLAP-relatedbiallelic marker is selected from the group consisting of A11 to A13,A15, A17 to A21, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith; optionally,wherein said FLAP-related biallelic marker is either A14 or A16, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, wherein the 3′ end of saidpolynucleotide is located 1 nucleotide upstream of said FLAP-relatedbiallelic marker in said sequence; and optionally, wherein saidpolynucleotide consists essentially of a sequence selected from thefollowing sequences: D1 to D28 and E1 to E28; and optionally, whereinsaid polynucleotide consists essentially of a sequence selected from thefollowing sequences: D1 to D13, D15, D17 to D28, E1 to E13, E15, and E17to E28. In a further embodiment, the invention encompasses isolated,purified, or recombinant polynucleotides consisting of, or consistingessentially of a sequence selected from the following sequences: B1 toB17, and C1 to C17. To these primers can be added, at either endthereof, a further polynucleotide useful for sequencing. Preferably,primers PU contain the additional PU 5′ sequence of SEQ ID NO: 14 andprimers RP contain the RP 5′ sequence of SEQ ID NO: 15.

In an additional embodiment, the invention encompasses polynucleotidesfor use in hybridization assay sequencing assays, microsequencing assaysand enzyme-based mismatch detection assays for determining the identityof the nucleotide at a FLAP-related biallelic marker in SEQ ID NO: 1 or2, or the complement thereof, as well as polynucleotides for use inamplifying segments of nucleotides comprising a FLAP-related biallelicmarker in SEQ ID NO: 1 or 2, or the complement thereof, optionally,wherein said FLAP-related biallelic marker is selected from the groupconsisting of A1 to A28, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith; optionally fromthe group consisting of A1 to A13, A15, and A17 to A28, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally from the group consisting of A1 toA10 and A22 to A28, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith; optionally fromthe group consisting of A11 to A13, A15, A17 to A21, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally from the group consisting of A14 and A16, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith. Optionally, said polynucleotide may comprise asequence disclosed in the present specification; Optionally, saidpolynucleotide may consist of, or consist essentially of anypolynucleotide described in the present specification. A preferredpolynucleotide may be used in a hybridization assay for determining theidentity of the nucleotide at a biallelic marker of the FLAP gene.Another preferred polynucleotide may be used in a sequencing ormicrosequencing assay for determining the identity of the nucleotide ata biallelic marker of the FLAP gene. A third preferred polynucleotidemay be used in an enzyme-based mismatch detection assay for determiningthe identity of the nucleotide at a biallelic marker of the FLAP gene. Afourth preferred polynucleotide may be used in amplifying a segment ofpolynucleotides comprising a biallelic marker of the FLAP gene;Optionally, said amplifying may be performed by a PCR or LCR.Optionally, said polynucleotide may be attached to a solid support,array, or addressable array; Optionally, said polynucleotide may belabeled.

Primers and probes according to the invention are therefore synthesizedto be “substantially” complementary to a strand of the FLAP gene to beamplified. The primer sequence does not need to reflect the exactsequence of the DNA template. Minor mismatches can be accommodated byreducing the stringency of the hybridization conditions. Among thevarious methods available to design useful primers, the OSP computersoftware can be used by the skilled person (see Hillier & Green, 1991).

The formation of stable hybrids depends on the melting temperature (Tm)of the DNA. The Tm depends on the length of the primer or probe, theionic strength of the solution and the G+C content. The higher the G Ccontent of the primer or probe, the higher is the melting temperaturebecause G:C pairs are held by three H bonds whereas A:T pairs have onlytwo. The GC content in the primers and probes of the invention usuallyranges between 10 and 75%, preferably between 35 and 60%, and morepreferably between 40 and 55%.

Preferably, the length of the primer and probe can range from 10 to 100nucleotides, preferably from 10 to 50, 10 to 30 or more preferably 10 to25 nucleotides. Shorter primers and probes tend to lack specificity fora target nucleic acid sequence and generally require cooler temperaturesto form sufficiently stable hybrid complexes with the template. Longerprimers and probes are expensive to produce and can sometimesself-hybridize to form hairpin structures. The appropriate length forprimers and probes under a particular set of assay conditions may beempirically determined by one of skill in the art.

The probes of the present invention are useful for a number of purposes.They can be used in Southern hybridization to genomic DNA or Northernhybridization to mRNA. The probes can also be used to detect PCRamplification products. They may also be used to detect mismatches inthe FLAP gene or mRNA using other techniques. Generally, the probes arecomplementary to the FLAP gene coding sequences, although probes tointrons and regulatory sequences are also contemplated.

Primers and probes can be prepared by any suitable method, including,for example, cloning and restriction of appropriate sequences and directchemical synthesis by a method such as the phosphodiester method ofNarang et al. (1979), the phosphodiester method of Brown et al. (1979),the diethylphosphoramidite method of Beaucage et al. (1981) and thesolid support method described in EP 0 707 592. The disclosures of allthese documents are incorporated herein by reference.

Detection probes are generally nucleic acid sequences or unchargednucleic acid analogs such as, for example peptide nucleic acids whichare disclosed in International Patent Application WO 92/20702;morpholino analogs which are described in U.S. Pat. Nos. 5,185,444,5,034,506, and 5,142,047; and the like. The disclosures of each of thesepatents is incorporated herein by reference. Depending upon the type oflabel carried by the probe, the probe is employed to capture or detectthe amplicon generated by the amplification reaction. The probe is notinvolved in amplification of the target sequence and therefore may haveto be rendered “non-extendable” in that additional dNTPs cannot be addedto the probe. In and of themselves analogs usually are non-extendableand nucleic acid probes can be rendered non-extendable by modifying the3′ end of the probe such that the hydroxyl group is no longer capable ofparticipating in elongation. For example, the 3′ end of the probe can befunctionalized with the capture or detection label to thereby consume orotherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl groupsimply can be cleaved, replaced or modified. U.S. patent applicationSer. No. 07/049,061 filed Apr. 19, 1993 describes modifications whichcan be used render a probe non-extendable.

The probes are preferably directly labeled such as with isotopes,reporter molecules or fluorescent labels or indirectly labeled such aswith biotin to which a streptavidin complex may later bind. Probelabeling techniques are well-known to the skilled technician. Byassaying the presence of the probe, one can detect the presence orabsence of the targeted DNA sequence in a given sample. The same labelscan be used with primers. For example, useful labels include radioactivesubstances (³²P, ³⁵S, ³H, ¹²⁵I), fluorescent dyes (5-bromodesoxyuridin,fluorescein, acetylaminofluorene, digoxigenin). Preferably,polynucleotides are labeled at their 3′ and 5′ ends. Examples ofnon-radioactive labeling of nucleic acid fragments are described in theFrench patent No FR-7810975 or by Urdea et al. (1988) orSanchez-Pescador et al. (1988). In addition, the probes according to thepresent invention may have structural characteristics such that theyallow the signal amplification, such structural characteristics being,for example, branched DNA probes as those described by Urdea et al. in1991 or in the European patent No EP 0 225 807 (Chiron).

Any of the primers and probes of the present invention can beconveniently immobilized on a solid support. Solid supports are known tothose skilled in the art and include the walls of wells of a reactiontray, test tubes, polystyrene beads, magnetic beads, nitrocellulosestrips, membranes, microparticles such as latex particles, sheep (orother animal) red blood cells, duracytes and others. The “solid phase”is not critical and can be selected by one skilled in the art. Thus,latex particles, microparticles, magnetic or non-magnetic beads,membranes, plastic tubes, walls of microtiter wells, glass or siliconchips, sheep (or other suitable animal's) red blood cells and duracytesare all suitable examples.

Suitable methods for immobilizing nucleic acids on solid phases includeionic, hydrophobic, covalent interactions and the like. A “solid phase”,as used herein, refers to any material which is insoluble, or can bemade insoluble by a subsequent reaction. The solid phase can be chosenfor its intrinsic ability to attract and immobilize the capture reagent.

Alternatively, the solid phase can retain an additional receptor whichhas the ability to attract and immobilize the capture reagent. Theadditional receptor can include a charged substance that is oppositelycharged with respect to the capture reagent itself or to a chargedsubstance conjugated to the capture reagent.

As yet another alternative, the receptor molecule can be any specificbinding member which is immobilized upon (attached to) the solid phaseand which has the ability to immobilize the capture reagent through aspecific binding reaction. The receptor molecule enables the indirectbinding of the capture reagent to a solid phase material before theperformance of the assay or during the performance of the assay. Thesolid phase thus can be a plastic, derivatized plastic, magnetic ornon-magnetic metal, glass or silicon surface of a test tube, microtiterwell, sheet, bead, microparticle, chip, sheep (or other suitableanimal's) red blood cells, duracytes and other configurations known tothose of ordinary skill in the art.

The polynucleotides of the invention can be attached to or immobilizedon a solid support individually or in groups of at least 2, 5, 8, 10,12, 15, 20, or 25 distinct polynucleotides of the invention to a singlesolid support. In addition, polynucleotides other than those of theinvention may be attached to the same solid support as one or morepolynucleotides of the invention.

Consequently, the invention also deals with a method for detecting thepresence of a nucleic acid comprising a nucleotide sequence selectedfrom a group consisting of SEQ ID NOs: 1 and 2, a fragment or a variantthereof or a complementary sequence thereto in a sample, said methodcomprising the following steps of:

-   -   a) bringing into contact a nucleic acid probe or a plurality of        nucleic acid probes which can hybridize with a nucleotide        sequence included in a nucleic acid selected form the group        consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2, a        fragment or a variant thereof or a complementary sequence        thereto and the sample to be assayed.    -   b) detecting the hybrid complex formed between the probe and a        nucleic acid in the sample.

The invention further concerns a kit for detecting the presence of anucleic acid comprising a nucleotide sequence selected from a groupconsisting of SEQ ID NOs: 1 and 2, a fragment or a variant thereof or acomplementary sequence thereto in a sample, said kit comprising:

a) a nucleic acid probe or a plurality of nucleic acid probes which canhybridize with a nucleotide sequence included in a nucleic acid selectedform the group consisting of the nucleotide sequences of SEQ ID NOs: 1and 2, a fragment or a variant thereof or a complementary sequencethereto;

b) optionally, the reagents necessary for performing the hybridizationreaction.

In a first preferred embodiment of the detection method and kit, thenucleic acid probe or the plurality of nucleic acid probes are labeledwith a detectable molecule. In a second preferred embodiment of thedetection method and kit, the nucleic acid probe or the plurality ofnucleic acid probes has been immobilized on a substrate. In a thirdpreferred embodiment of the detection method and kit, the nucleic acidprobe or the plurality of nucleic acid probes comprise either a sequencewhich is selected from the group consisting of the nucleotide sequences:B1 to B17, C1 to C17, D1 to D28, E1 to E28, P1 to P28 or a biallelicmarker selected from the group consisting of A1 to A28 or thecomplements thereto or the biallelic markers in linkage disequilibriumtherewith.

Oligonucleotide Arrays

A substrate comprising a plurality of oligonucleotide primers or probesof the invention may be used either for detecting or amplifying targetedsequences in the FLAP gene and may also be used for detecting mutationsin the coding or in the non-coding sequences of the FLAP gene.

Any polynucleotide provided herein may be attached in overlapping areasor at random locations on the solid support. Alternatively thepolynucleotides of the invention may be attached in an ordered arraywherein each polynucleotide is attached to a distinct region of thesolid support which does not overlap with the attachment site of anyother polynucleotide. Preferably, such an ordered array ofpolynucleotides is designed to be “addressable” where the distinctlocations are recorded and can be accessed as part of an assayprocedure. Addressable polynucleotide arrays typically comprise aplurality of different oligonucleotide probes that are coupled to asurface of a substrate in different known locations. The knowledge ofthe precise location of each polynucleotides location makes these“addressable” arrays particularly useful in hybridization assays. Anyaddressable array technology known in the art can be employed with thepolynucleotides of the invention. One particular embodiment of thesepolynucleotide arrays is known as the Genechips™, and has been generallydescribed in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and92/10092. These arrays may generally be produced using mechanicalsynthesis methods or light directed synthesis methods which incorporatea combination of photolithographic methods and solid phaseoligonucleotide synthesis (Fodor et al., 1991). The immobilization ofarrays of oligonucleotides on solid supports has been rendered possibleby the development of a technology generally identified as “Very LargeScale Immobilized Polymer Synthesis” (VLSIPS™) in which, typically,probes are immobilized in a high density array on a solid surface of achip. Examples of VLSIPS™ technologies are provided in U.S. Pat. Nos.5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092and WO 95/11995, which describe methods for forming oligonucleotidearrays through techniques such as light-directed synthesis techniques.In designing strategies aimed at providing arrays of nucleotidesimmobilized on solid supports, further presentation strategies weredeveloped to order and display the oligonucleotide arrays on the chipsin an attempt to maximize hybridization patterns and sequenceinformation. Examples of such presentation strategies are disclosed inPCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256.

In another embodiment of the oligonucleotide arrays of the invention, anoligonucleotide probe matrix may advantageously be used to detectmutations occurring in the FLAP gene. For this particular purpose,probes are specifically designed to have a nucleotide sequence allowingtheir hybridization to the genes that carry known mutations (either bydeletion, insertion or substitution of one or several nucleotides). Byknown mutations, it is meant, mutations on the FLAP gene that have beenidentified according, for example to the technique used by Huang et al.(1996) or Samson et al. (1996).

Another technique that is used to detect mutations in the FLAP gene isthe use of a high-density DNA array. Each oligonucleotide probeconstituting a unit element of the high density DNA array is designed tomatch a specific subsequence of the FLAP genomic DNA or cDNA. Thus, anarray consisting of oligonucleotides complementary to subsequences ofthe target gene sequence is used to determine the identity of the targetsequence with the wild gene sequence, measure its amount, and detectdifferences between the target sequence and the reference wild genesequence of the FLAP gene. In one such design, termed 4L tiled array, isimplemented a set of four probes (A, C, G, T), preferably 15-nucleotideoligomers. In each set of four probes, the perfect complement willhybridize more strongly than mismatched probes. Consequently, a nucleicacid target of length L is scanned for mutations with a tiled arraycontaining 4L probes, the whole probe set containing all the possiblemutations in the known wild reference sequence. The hybridizationsignals of the 15-mer probe set tiled array are perturbed by a singlebase change in the target sequence. As a consequence, there is acharacteristic loss of signal or a “footprint” for the probes flanking amutation position. This technique was described by Chee et al. in 1996,which is herein incorporated by reference.

Consequently, the invention concerns an array of nucleic acid moleculescomprising at least one polynucleotide described above as probes andprimers. Preferably, the invention concerns an array of nucleic acidcomprising at least two polynucleotides described above as probes andprimers.

A further object of the invention consists of an array of nucleic acidsequences comprising either at least one of the sequences selected fromthe group consisting of P1 to P28, B1 to B17, C1 to C17, D1 to D28 andE1 to E28 or the sequences complementary thereto or a fragment thereofof at least 8, 10, 12, 15, 18, 20, 25, 30, or 40 consecutive nucleotidesthereof, or at least one sequence comprising a biallelic marker selectedfrom the group consisting of A1 to A28, and the complements thereto, oroptionally the biallelic markers in linkage disequilibrium therewith.

The invention also pertains to an array of nucleic acid sequencescomprising either at least two of the sequences selected from the groupconsisting of P1 to P28, B1 to B17, C1 to C17, D1 to D28 and E1 to E28or the sequences complementary thereto or a fragment thereof of at least8 consecutive nucleotides thereof, or at least two sequences comprisinga biallelic marker selected from the group consisting of A1 to A28, andthe complements thereto, or optionally the biallelic markers in linkagedisequilibrium therewith.

VII. Identification of Biallelic Markers

There are two preferred methods through which the biallelic markers ofthe present invention can be generated. In a first method, DNA samplesfrom unrelated individuals are pooled together, following which thegenomic DNA of interest is amplified and sequenced. The nucleotidesequences thus obtained are then analyzed to identify significantpolymorphisms.

One of the major advantages of this method resides in the fact that thepooling of the DNA samples substantially reduces the number of DNAamplification reactions and sequencing reactions which must be carriedout. Moreover, this method is sufficiently sensitive so that a biallelicmarker obtained therewith usually shows a sufficient degree ofinformativeness for conducting association studies.

In a second method for generating biallelic markers, the DNA samples arenot pooled and are therefore amplified and sequenced individually. Theresulting nucleotide sequences obtained are then also analyzed toidentify significant polymorphisms.

It will readily be appreciated that when this second method is used, asubstantially higher number of DNA amplification reactions andsequencing reactions must be carried out. Moreover, a biallelic markerobtained using this method may show a lower degree of informativenessfor conducting association studies, e.g. if the frequency of its lessfrequent allele may be less than about 10%. It will further beappreciated that including such less informative biallelic markers inassociation studies to identify potential genetic associations with atrait may allow in some cases the direct identification of causalmutations, which may, depending on their penetrance, be rare mutations.This method is usually preferred when biallelic markers need to beidentified in order to perform association studies within candidategenes.

The following is a description of the various parameters of a preferredmethod used by the inventors to generate the markers of the presentinvention.

1. DNA Extraction

The genomic DNA samples from which the biallelic markers of the presentinvention are generated are preferably obtained from unrelatedindividuals corresponding to a heterogeneous population of known ethnicbackground.

The number of individuals from whom DNA samples are obtained can varysubstantially, preferably from about 10 to about 1000, preferably fromabout 50 to about 200 individuals. It is usually preferred to collectDNA samples from at least about 100 individuals in order to havesufficient polymorphic diversity in a given population to identify asmany markers as possible and to generate statistically significantresults.

As for the source of the genomic DNA to be subjected to analysis, anytest sample can be foreseen without any particular limitation. Thesetest samples include biological samples which can be tested by themethods of the present invention described herein and include human andanimal body fluids such as whole blood, serum, plasma, cerebrospinalfluid, urine, lymph fluids, and various external secretions of therespiratory, intestinal and genitourinary tracts, tears, saliva, milk,white blood cells, myelomas and the like; biological fluids such as cellculture supernatants; fixed tissue specimens including tumor andnon-tumor tissue and lymph node tissues; bone marrow aspirates and fixedcell specimens. The preferred source of genomic DNA used in the contextof the present invention is from peripheral venous blood of each donor.

The techniques of DNA extraction are well-known to the skilledtechnician. Details of a preferred embodiment are provided in Example 1.

Once genomic DNA from every individual in the given population has beenextracted, it is preferred that a fraction of each DNA sample isseparated, after which a pool of DNA is constituted by assemblingequivalent amounts of the separated fractions into a single one.However, the person skilled in the art can choose to amplify the pooledor unpooled sequences

2. DNA Amplification

The identification of biallelic markers in a sample of genomic DNA maybe facilitated through the use of DNA amplification methods. DNA samplescan be pooled or unpooled for the amplification step.

DNA amplification techniques are well-known to those skilled in the art.Amplification techniques that can be used in the context of the presentinvention include, but are not limited to, the ligase chain reaction(LCR) described in EP-A-320 308, WO 9320227 and EP-A-439 182, thedisclosures of which are incorporated herein by reference, the polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleicacid sequence based amplification (NASBA) described in Guatelli J. C.,et al. (1990) and in Compton J. (1991), Q-beta amplification asdescribed in European Patent Application No 454-4610, stranddisplacement amplification as described in Walker et al. (1996) and EP A684 315 and, target mediated amplification as described in PCTPublication WO 9322461, the disclosures of which are incorporated hereinby reference.

LCR and Gap LCR are exponential amplification techniques, both depend onDNA ligase to join adjacent primers annealed to a DNA molecule. InLigase Chain Reaction (LCR), probe pairs are used which include twoprimary (first and second) and two secondary (third and fourth) probes,all of which are employed in molar excess to target. The first probehybridizes to a first segment of the target strand and the second probehybridizes to a second segment of the target strand, the first andsecond segments being contiguous so that the primary probes abut oneanother in 5′ phosphate-3′hydroxyl relationship, and so that a ligasecan covalently fuse or ligate the two probes into a fused product. Inaddition, a third (secondary) probe can hybridize to a portion of thefirst probe and a fourth (secondary) probe can hybridize to a portion ofthe second probe in a similar abutting fashion. Of course, if the targetis initially double stranded, the secondary probes also will hybridizeto the target complement in the first instance. Once the ligated strandof primary probes is separated from the target strand, it will hybridizewith the third and fourth probes, which can be ligated to form acomplementary, secondary ligated product. It is important to realizethat the ligated products are functionally equivalent to either thetarget or its complement. By repeated cycles of hybridization andligation, amplification of the target sequence is achieved. A method formultiplex LCR has also been described (WO 9320227). Gap LCR (GLCR) is aversion of LCR where the probes are not adjacent but are separated by 2to 3 bases.

For amplification of mRNAs, it is within the scope of the presentinvention to reverse transcribe mRNA into cDNA followed by polymerasechain reaction (RT-PCR); or, to use a single enzyme for both steps asdescribed in U.S. Pat. No 5,322,770 or, to use Asymmetric Gap LCR(RT-AGLCR) as described by Marshall et al. (1994). AGLCR is amodification of GLCR that allows the amplification of RNA.

The PCR technology is the preferred amplification technique used in thepresent invention. A variety of PCR techniques are familiar to thoseskilled in the art. For a review of PCR technology, see White (1997) andthe publication entitled “PCR Methods and Applications” (1991, ColdSpring Harbor Laboratory Press). In each of these PCR procedures, PCRprimers on either side of the nucleic acid sequences to be amplified areadded to a suitably prepared nucleic acid sample along with dNTPs and athermostable polymerase such as Taq polymerase, Pfu polymerase, or Ventpolymerase. The nucleic acid in the sample is denatured and the PCRprimers are specifically hybridized to complementary nucleic acidsequences in the sample. The hybridized primers are extended.Thereafter, another cycle of denaturation, hybridization, and extensionis initiated. The cycles are repeated multiple times to produce anamplified fragment containing the nucleic acid sequence between theprimer sites. PCR has further been described in several patentsincluding U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188. Each ofthese publications is incorporated herein by reference.

The PCR technology is the preferred amplification technique used toidentify new biallelic markers. A typical example of a PCR reactionsuitable for the purposes of the present invention is provided inExample 2.

One of the aspects of the present invention is a method for theamplification of the human FLAP gene, particularly of the genomicsequence of SEQ ID NO: 1 or of the cDNA sequence of SEQ ID NO: 2, or afragment or a variant thereof in a test sample, preferably using the PCRtechnology. This method comprises the steps of contacting a test samplesuspected of containing the target FLAP encoding sequence or portionthereof with amplification reaction reagents comprising a pair ofamplification primers, and eventually in some instances a detectionprobe that can hybridize with an internal region of amplicon sequencesto confirm that the desired amplification reaction has taken place.

Thus, the present invention also relates to a method for theamplification of a human FLAP gene sequence, particularly of a portionof the genomic sequences of SEQ ID NO: 1 or of the cDNA sequence of SEQID NO: 2, or a variant thereof in a test sample, said method comprisingthe steps of:

-   -   a) contacting a test sample suspected of containing the targeted        FLAP gene sequence comprised in a nucleotide sequence selected        from a group consisting of SEQ ID NOs: 1 and 2, or fragments or        variants thereof with amplification reaction reagents comprising        a pair of amplification primers as described above and located        on either side of the polynucleotide region to be amplified, and    -   b) optionally, detecting the amplification products.

The invention also concerns a kit for the amplification of a human FLAPgene sequence, particularly of a portion of the genomic sequence of SEQID NO: 1 or of the cDNA sequence of SEQ ID NO: 2, or a variant thereofin a test sample, wherein said kit comprises:

a) a pair of oligonucleotide primers located on either side of the FLAPregion to be amplified;

b) optionally, the reagents necessary for performing the amplificationreaction.

In one embodiment of the above amplification method and kit, theamplification product is detected by hybridization with a labeled probehaving a sequence which is complementary to the amplified region. Inanother embodiment of the above amplification method and kit, primerscomprise a sequence which is selected from the group consisting of thenucleotide sequences of B1 to B17, C11 to C17, D1 to D28 and E1 to E28.In a preferred embodiment of the above amplification method and kit, theamplification product comprises a polymorphic base of a biallelic markerof the present invention, more particularly a polymorphic base of abiallelic marker selected from the group of A1 to A28, optionally fromthe group consisting of A1 to A13, A15 and A17 to A28, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith. The primers are more particularlycharacterized in that they have sufficient complementarity with anysequence of a strand of the genomic sequence close to the region to beamplified, for example with a non-coding sequence adjacent to exons toamplify.

In a first embodiment of the present invention, biallelic markers areidentified using genomic sequence information generated by theinventors. Sequenced genomic DNA fragments are used to design primersfor the amplification of 500 bp fragments. These 500 bp fragments areamplified from genomic DNA and are scanned for biallelic markers.Primers may be designed using the OSP software (Hillier L. and Green P.,1991). All primers may contain, upstream of the specific target bases, acommon oligonucleotide tail that serves as a sequencing primer. Thoseskilled in the art are familiar with primer extensions, which can beused for these purposes.

Preferred primers, useful for the amplification of genomic sequencesencoding the candidate genes, focus on promoters, exons and splice sitesof the genes. A biallelic marker presents a higher probability to be aneventual causal mutation if it is located in these functional regions ofthe gene. Preferred amplification primers of the invention include thenucleotide sequences B1 to B17 and the nucleotide sequences C1 to C17disclosed in Example 2.

3. Sequencing of Amplified Genomic DNA and Identification ofPolymorphisms

The amplification products generated as described above with the primersof the invention are then sequenced using methods known and available tothe skilled technician. Preferably, the amplified DNA is subjected toautomated dideoxy terminator sequencing reactions using a dye-primercycle sequencing protocol.

Following gel image analysis and DNA sequence extraction, sequence dataare automatically processed with software to assess sequence quality.

The sequence data obtained as described above are subjected to qualitycontrol and validation steps based on the shape of the peak, theinter-peak resolution, the number of unreliable peaks in a particularstretch of sequence and the noise level. Sequence data that isconsidered unreliable is discarded.

After this first sequence quality analysis, polymorphisms are detectedamong individual or pooled amplified fragment sequences. Thepolymorphism search is based on the presence of superimposed peaks inthe electrophoresis pattern. These peaks, which present two distinctcolors, correspond to two different nucleotides at the same position onthe sequence. In order for peaks to be considered significant, peakheight has to satisfy conditions of ratio between the peaks andconditions of ratio between a given peak and the surrounding peaks ofthe same color.

However, since the presence of two peaks can be an artifact due tobackground noise, two controls are utilized to exclude these artifacts:

-   -   the two DNA strands are sequenced and a comparison between the        peaks is carried out. The polymorphism has to be detected on        both strands for validation.    -   all the sequencing electrophoresis patterns of the same        amplification product provided from distinct pools and/or        individuals are compared. The homogeneity and the ratio of        homozygous and heterozygous peak height are controlled through        these distinct DNAs.

The detection limit for the frequency of biallelic polymorphismsdetected by sequencing pools of 100 individuals is about 0.1 for theminor allele, as verified by sequencing pools of known allelicfrequencies. However, more than 90% of the biallelic polymorphismsdetected by the pooling method have a frequency for the minor allelehigher than 0.25. Therefore, the biallelic markers selected by thismethod have a frequency of at least 0.1 for the minor allele and lessthan 0.9 for the major allele, preferably at least 0.2 for the minorallele and less than 0.8 for the major allele, more preferably at least0.3 for the minor allele and less than 0.7 for the major allele, thus aheterozygosity rate higher than 0.18, preferably higher than 0.32, morepreferably higher than 0.42.

In another embodiment, biallelic markers are detected by sequencingindividual DNA samples, the frequency of the minor allele of such abiallelic marker may be less than 0.1.

4. Validation of the Biallelic Markers of the Present Invention

The polymorphisms are evaluated for their usefulness as genetic markersby validating that both alleles are present in a population. Validationof the biallelic markers is accomplished by genotyping a group ofindividuals by a method of the invention and demonstrating that bothalleles are present. Microsequencing is a preferred method of genotypingalleles. The validation by genotyping step may be performed onindividual samples derived from each individual in the group or bygenotyping a pooled sample derived from more than one individual. Thegroup can be as small as one individual if that individual isheterozygous for the allele in question. Preferably the group containsat least three individuals, more preferably the group contains five orsix individuals, so that a single validation test will be more likely toresult in the validation of more of the biallelic markers that are beingtested. It should be noted, however, that when the validation test isperformed on a small group it may result in a false negative result ifas a result of sampling error none of the individuals tested carries oneof the two alleles. Thus, the validation process is less useful indemonstrating that a particular initial result is an artifact, than itis at demonstrating that there is a bona fide biallelic marker at aparticular position in a sequence.

5. Evaluation of the Frequency of the Biallelic Markers of the PresentInvention

The validated biallelic markers are further evaluated for theirusefulness as genetic markers by determining the frequency of the leastcommon allele at the biallelic marker site. The higher the frequency ofthe less common allele the greater the usefulness of the biallelicmarker is in association studies. The determination of the least commonallele is accomplished by genotyping a group of individuals by a methodof the invention and demonstrating that both alleles are present. Thisdetermination of frequency by genotyping step may be performed onindividual samples derived from each individual in the group or bygenotyping a pooled sample derived from more than one individual. Thegroup must be large enough to be representative of the population as awhole. Preferably the group contains at least 20 individuals, morepreferably the group contains at least 50 individuals, most preferablythe group contains at least 100 individuals. Of course the larger thegroup the greater the accuracy of the frequency determination because ofreduced sampling error. For an indication of the frequency for the lesscommon allele of a particular biallelic marker of the invention seeTable 2. A biallelic marker wherein the frequency of the less commonallele is 30% or more is termed a “high quality biallelic marker.”

The invention also relates to methods of estimating the frequency of anallele of a FLAP-related biallelic marker in a population comprising: a)genotyping individuals from said population for said biallelic markeraccording to the method of the present invention; b) determining theproportional representation of said biallelic marker in said population.In addition, the methods of estimating the frequency of an allele in apopulation of the invention encompass methods with any furtherlimitation described in this disclosure, or those following, specifiedalone or in any combination; Optionally, said FLAP-related biallelicmarker may be selected from the group consisting of A1 to A28, and thecomplements thereof; Optionally, said FLAP-related biallelic marker maybe selected from the group consisting of A1 to A13, A15, and A17 to A28,and the complements thereof, or optionally the biallelic markers inlinkage disequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A1 to A10 and A22 to A28, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A11 to A13, A15, A17 to A21, andthe complements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A14 or A16, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; Optionally, determining the proportional representation of anucleotide at a FLAP-related biallelic marker may be accomplished bydetermining the identity of the nucleotides for both copies of saidbiallelic marker present in the genome of each individual in saidpopulation and calculating the proportional representation of saidnucleotide at said FLAP-related biallelic marker for the population;Optionally, determining the proportional representation may beaccomplished by performing a genotyping method of the invention on apooled biological sample derived from a representative number ofindividuals, or each individual, in said population, and calculating theproportional amount of said nucleotide compared with the total.

VIII. Methods for Genotyping an Individual for Biallelic Markers

Methods are provided to genotype a biological sample for one or morebiallelic markers of the present invention, all of which may beperformed in vitro. Such methods of genotyping comprise determining theidentity of a nucleotide at a FLAP biallelic marker site by any methodknown in the art. These methods find use in genotyping case-controlpopulations in association studies as well as individuals in the contextof detection of alleles of biallelic markers which are known to beassociated with a given trait, in which case both copies of thebiallelic marker present in individual's genome are determined so thatan individual may be classified as homozygous or heterozygous for aparticular allele.

These genotyping methods can be performed on nucleic acid samplesderived from a single individual or pooled DNA samples.

The identification of biallelic markers described previously allows thedesign of appropriate oligonucleotides, which can be used as probes andprimers, to amplify a FLAP gene containing the polymorphic site ofinterest and for the detection of such polymorphisms.

Genotyping can be performed using similar methods as those describedabove for the identification of the biallelic markers, or using othergenotyping methods such as those further described below. In preferredembodiments, the comparison of sequences of amplified genomic fragmentsfrom different individuals is used to identify new biallelic markerswhereas microsequencing is used for genotyping known biallelic markersin diagnostic and association study applications.

The invention also pertains to a method of genotyping comprisingdetermining the identity of a nucleotide at a biallelic marker of theFLAP gene in a biological sample. Optionally, the biological sample isderived from a single subject; Optionally, the identity of thenucleotides at said biallelic marker is determined for both copies ofsaid biallelic marker present in said individual's genome. Optionally,said method is performed in vitro; Optionally, the biological sample isderived from multiple subjects. Optionally, the method of genotypingdescribed above further comprises amplifying a portion of said sequencecomprising the biallelic marker prior to said determining step;Optionally, wherein said amplifying is performed by PCR, LCR, orreplication of a recombinant vector comprising an origin of replicationand said portion in a host cell. The determining step of the abovegenotyping method may be performed either by a hybridization assay, asequencing assay, an enzyme-based mismatch detection assay and by amicrosequencing assay. Thus, the invention also encompasses methods ofgenotyping a biological sample comprising determining the identity of anucleotide at a FLAP-related biallelic marker. In addition, thegenotyping methods of the invention encompass methods with any furtherlimitation described in this disclosure, or those following, specifiedalone or in any combination. Optionally, said biallelic marker isselected from the group consisting of A1 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, said biallelic marker is selected from the groupconsisting of A1 to A13, A15 and A17 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, said biallelic marker is selected from the groupconsisting of A1 to A10 and A22 to A28, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith;optionally, said biallelic marker is selected from the group consistingof A11 to A13, A15, A17 to A21, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith;optionally, said biallelic marker is either A14 or A16, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith.

Source of DNA for Genotyping

Any source of nucleic acids, in purified or non-purified form, can beutilized as the starting nucleic acid, provided it contains or issuspected of containing the specific nucleic acid sequence desired. DNAor RNA may be extracted from cells, tissues, body fluids and the like asdescribed above. While nucleic acids for use in the genotyping methodsof the invention can be derived from any mammalian source, the testsubjects and individuals from which nucleic acid samples are taken aregenerally understood to be human.

Amplification of DNA Fragments Comprising Biallelic Markers

Methods and polynucleotides are provided to amplify a segment ofnucleotides comprising one or more biallelic marker of the presentinvention. It will be appreciated that amplification of DNA fragmentscomprising biallelic markers may be used in various methods and forvarious purposes and is not restricted to genotyping. Nevertheless, manygenotyping methods, although not all, require the previous amplificationof the DNA region carrying the biallelic marker of interest. Suchmethods specifically increase the concentration or total number ofsequences that span the biallelic marker or include that site andsequences located either distal or proximal to it. Diagnostic assays mayalso rely on amplification of DNA segments carrying a biallelic markerof the present invention. Amplification of DNA may be achieved by anymethod known in the art. Amplification techniques are described above inthe section entitled, “Identification of biallelic markers” VII. (2).

Some of these amplification methods are particularly suited for thedetection of single nucleotide polymorphisms and allow the simultaneousamplification of a target sequence and the identification of thepolymorphic nucleotide as it is further described below.

The identification of biallelic markers as described above allows thedesign of appropriate oligonucleotides, which can be used as primers toamplify DNA fragments comprising the biallelic markers of the presentinvention. Amplification can be performed using the primers initiallyused to discover new biallelic markers which are described herein or anyset of primers allowing the amplification of a DNA fragment comprising abiallelic marker of the present invention.

In some embodiments the present invention provides primers foramplifying a DNA fragment containing one or more biallelic markers ofthe present invention. In some embodiments, the primer pair is adaptedfor amplifying a sequence containing the polymorphic base of one of thesequences of P1 to P28, optionally P1 to P13, P15, P17 to P28, and thecomplementary sequence thereto. Preferred amplification primers arelisted in Example 2. It will be appreciated that the primers listed aremerely exemplary and that any other set of primers which produceamplification products containing one or more biallelic markers of thepresent invention.

The spacing of the primers determines the length of the segment to beamplified. In the context of the present invention, amplified segmentscarrying biallelic markers can range in size from at least about 25 hpto 35 kbp. Amplification fragments from 25-3000 bp are typical,fragments from 50-1000 bp are preferred and fragments from 100-600 bpare highly preferred. In a preferred embodiment of the invention, thepairs of primers for amplification and sequencing are sufficientlycomplementary with a region of a FLAP gene located at less than 500 bp,preferably at less than 100 bp, and more preferably at less than 50 bpof a polymorphic site corresponding to one of the markers of the presentinvention. Amplification primers may be labeled or immobilized on asolid support as described in “Oligonucleotide probes and primers”.

Methods of Genotyping DNA Samples for Biallelic Markers

Any method known in the art can be used to identify the nucleotidepresent at a biallelic marker site. Since the biallelic marker allele tobe detected has been identified and specified in the present invention,detection will prove simple for one of ordinary skill in the art byemploying any of a number of techniques. Many genotyping methods requirethe previous amplification of the DNA region carrying the biallelicmarker of interest. While the amplification of target or signal is oftenpreferred at present, ultrasensitive detection methods which do notrequire amplification are also encompassed by the present genotypingmethods. Methods well-known to those skilled in the art that can be usedto detect biallelic polymorphisms include methods such as, conventionaldot blot analyzes, single strand conformational polymorphism analysis(SSCP) described by Orita et al. (1989), denaturing gradient gelelectrophoresis (DGGE), heteroduplex analysis, mismatch cleavagedetection, and other conventional techniques as described in Sheffieldet al. (1991), White et al. (1992), Grompe et al. (1989 and 1993).Another method for determining the identity of the nucleotide present ata particular polymorphic site employs a specializedexonuclease-resistant nucleotide derivative as described in U.S. Pat.No. 4,656,127.

Preferred methods involve directly determining the identity of thenucleotide present at a biallelic marker site by a sequencing assay, anenzyme-based mismatch detection assay, or a hybridization assay. Thefollowing is a description of some preferred methods. A highly preferredmethod is the microsequencing technique. The term “sequencing” is usedherein to refer to polymerase extension of duplex primer/templatecomplexes and includes both traditional sequencing and microsequencing.

1) Sequencing Assays

The amplification products generated above with the primers of theinvention can be sequenced using methods known and available to theskilled technician. Preferably, the amplified DNA is subjected toautomated dideoxy terminator sequencing reactions using a dye-primercycle sequencing protocol. A sequence analysis can allow theidentification of the base present at the polymorphic site.

2) Microsequencing Assays

Polymorphism analyses on pools or selected individuals of a givenpopulation can be carried out by conducting microsequencing reactions oncandidate regions contained in amplified fragments obtained by PCRperformed on DNA or RNA samples taken from these individuals.

To do so, DNA samples are subjected to PCR amplification of thecandidate regions under conditions similar to those described above.These genomic amplification products are then subjected to automatedmicrosequencing reactions using ddNTPs (specific fluorescence for eachddNTP) and appropriate oligonucleotide microsequencing primers which canhybridize just upstream of the polymorphic base of interest. Oncespecifically extended at the 3′ end by a DNA polymerase using acomplementary fluorescent dideoxynucleotide analog (thermal cycling),the primer is precipitated to remove the unincorporated fluorescentddNTPs. The reaction products in which fluorescent ddNTPs have beenincorporated are then analyzed by electrophoresis on ABI 377 sequencingmachines to determine the identity of the incorporated base, therebyidentifying the polymorphic marker present in the sample.

An example of a typical microsequencing procedure that can be used inthe context of the present invention is provided in example 4. It is tobe understood that certain parameters of this procedure such as theelectrophoresis method or the labeling of ddNTPs could be modified bythe skilled person without substantially modifying its result.

The extended primer may also be analyzed by MALDI-TOF Mass Spectrometry.The base at the polymorphic site is identified by the mass added ontothe microsequencing primer (see Haff and Smirnov, 1997).

As a further alternative to the process described above, several solidphase microsequencing reactions have been developed. The basicmicrosequencing protocol is the same as described previously, exceptthat either the oligonucleotide microsequencing primers or thePCR-amplified products of the DNA fragment of interest are immobilized.For example, immobilization can be carried out via an interactionbetween biotinylated DNA and streptavidin-coated microtitration wells oravidin-coated polystyrene particles.

In such solid phase microsequencing reactions, incorporated ddNTPs caneither be radiolabeled (see Syvänen, 1994, incorporated herein byreference) or linked to fluorescein (see Livak & Hainer, 1994,incorporated herein by reference). The detection of radiolabeled ddNTPscan be achieved through scintillation-based techniques. The detection offluorescein-linked ddNTPs can be based on the binding of antifluoresceinantibody conjugated with alkaline phosphatase, followed by incubationwith a chromogenic substrate (such asp-nitrophenyl phosphate).

Other possible of reporter-detection couples include: ddNTP linked todinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (seeHarju et al., 1993, incorporated herein by reference); and biotinylatedddNTP and horseradish peroxidase-conjugated streptavidin witho-phenylenediamine as a substrate (see WO 92/115712, incorporated hereinby reference).

A diagnosis kit based on fluorescein-linked ddNTP with antifluoresceinantibody conjugated with alkaline phosphatase is commercialized underthe name PRONTO by GamidaGen Ltd.

As yet another alternative microsequencing procedure, Nyren et al.(1993) presented a concept of solid-phase DNA sequencing that relies onthe detection of DNA polymerase activity by an enzymatic luminometricinorganic pyrophosphate detection assay (ELIDA). The PCR-amplifiedproducts are biotinylated and immobilized on beads. The microsequencingprimer is annealed and four aliquots of this mixture are separatelyincubated with DNA polymerase and one of the four different ddNTPs.After the reaction, the resulting fragments are washed and used assubstrates in a primer extension reaction with all four dNTPs present.The progress of the DNA-directed polymerization reactions are monitoredwith the ELIDA. Incorporation of a ddNTP in the first reaction preventsthe formation of pyrophosphate during the subsequent dNTP reaction. Incontrast, no ddNTP incorporation in the first reaction gives extensivepyrophosphate release during the dNTP reaction and this leads togeneration of light throughout the ELIDA reactions. From the ELIDAresults, the first base after the primer is easily deduced.

Pastinen et al. (1997) describe a method for multiplex detection ofsingle nucleotide polymorphism in which the solid phase minisequencingprinciple is applied to an oligonucleotide array format. High-densityarrays of DNA probes attached to a solid support (DNA chips) are furtherdescribed below.

In one aspect the present invention provides polynucleotides and methodsto genotype one or more biallelic markers of the present invention byperforming a microsequencing assay. Preferably, the biallelic markersare selected from the group consisting of A1 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith. Optionally, the biallelic markers are selected from the groupconsisting of A1 to A13, A15, A17 to A28, and the complements thereof,or optionally the biallelic markers in linkage disequilibrium therewith.Preferred microsequencing primers include the nucleotide sequences: D1to D28 and E1 to E28. Optionally, microsequencing primers include thenucleotide sequences: D1 to D13, D15, D17 to D28, E1 to E13, E15, andE17 to E28. More preferred microsequencing primers are selected from thegroup consisting of the nucleotide sequences: E11, D12, D13, D14, D15,D16, E18, D19, and E20. It will be appreciated that the microsequencingprimers listed in Example 4 are merely exemplary and that, any primerhaving a 3′ end immediately adjacent to the polymorphic nucleotide maybe used. Similarly, it will be appreciated that microsequencing analysismay be performed for any biallelic marker or any combination ofbiallelic markers of the present invention. One aspect of the presentinvention is a solid support which includes one or more microsequencingprimers listed in Example 4, or fragments comprising at least 8, 12, 15,20, 25, 30, 40, or 50 consecutive nucleotides thereof and having a 3′terminus immediately upstream of the corresponding biallelic marker, fordetermining the identity of a nucleotide at a biallelic marker site.

3) Mismatch Detection Assays Based on Polymerases and Ligases

In one aspect the present invention provides polynucleotides and methodsto determine the allele of one or more biallelic markers of the presentinvention in a biological sample, by allele-specific amplificationassays. Methods, primers and various parameters to amplify DNA fragmentscomprising biallelic markers of the present invention are furtherdescribed above in “Amplification Of DNA Fragments Comprising BiallelicMarkers”.

Allele Specific Amplification Primers

Discrimination between the two alleles of a biallelic marker can also beachieved by allele specific amplification, a selective strategy, wherebyone of the alleles is amplified without amplification of the otherallele. For allele specific amplification, at least one member of thepair of primers is sufficiently complementary with a region of a FLAPgene comprising the polymorphic base of a biallelic marker of thepresent invention to hybridize therewith. Such primers are able todiscriminate between the two alleles of a biallelic marker.

This can be accomplished by placing the polymorphic base at the 3′ endof one of the amplification primers. Such allele specific primers tendto selectively prime an amplification or sequencing reaction so long asthey are used with a nucleic acid sample that contains one of the twoalleles present at a biallelic marker because the extension forms fromthe 3′ end of the primer, a mismatch at or near this position has aninhibitory effect on amplification. Therefore, under appropriateamplification conditions, these primers only direct amplification ontheir complementary allele. Determining the precise location of themismatch and the corresponding assay conditions are well with theordinary skill in the art.

Ligation/Amplification Based Methods

The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotideswhich are designed to be capable of hybridizing to abutting sequences ofa single strand of a target molecules. One of the oligonucleotides isbiotinylated, and the other is detectably labeled. If the precisecomplementary sequence is found in a target molecule, theoligonucleotides will hybridize such that their termini abut, and createa ligation substrate that can be captured and detected. OLA is capableof detecting single nucleotide polymorphisms and may be advantageouslycombined with PCR as described by Nickerson et al. (1990). In thismethod, PCR is used to achieve the exponential amplification of targetDNA, which is then detected using OLA.

Other amplification methods which are particularly suited for thedetection of single nucleotide polymorphism include LCR (ligase chainreaction), Gap LCR (GLCR) which are described above in “IdentificationOf Biallelic Markers” (2). LCR uses two pairs of probes to exponentiallyamplify a specific target. The sequences of each pair ofoligonucleotides, is selected to permit the pair to hybridize toabutting sequences of the same strand of the target. Such hybridizationforms a substrate for a template-dependant ligase. In accordance withthe present invention, LCR can be performed with oligonucleotides havingthe proximal and distal sequences of the same strand of a biallelicmarker site. In one embodiment, either oligonucleotide will be designedto include the biallelic marker site. In such an embodiment, thereaction conditions are selected such that the oligonucleotides can beligated together only if the target molecule either contains or lacksthe specific nucleotide that is complementary to the biallelic marker onthe oligonucleotide. In an alternative embodiment, the oligonucleotideswill not include the biallelic marker, such that when they hybridize tothe target molecule, a “gap” is created as described in WO 90/01069.This gap is then “filled” with complementary dNTPs (as mediated by DNApolymerase), or by an additional pair of oligonucleotides. Thus at theend of each cycle, each single strand has a complement capable ofserving as a target during the next cycle and exponentialallele-specific amplification of the desired sequence is obtained.

Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method fordetermining the identity of a nucleotide at a preselected site in anucleic acid molecule (WO 95/21271). This method involves theincorporation of a nucleoside triphosphate that is complementary to thenucleotide present at the preselected site onto the terminus of a primermolecule, and their subsequent ligation to a second oligonucleotide. Thereaction is monitored by detecting a specific label attached to thereaction's solid phase or by detection in solution.

4) Hybridization Assay Methods

The invention also relates to a group of probes characterized in thatthey preferably comprise between 10 and 50 nucleotides, and in that theyare sufficiently complementary to a polymorphic sequence defined by abiallelic marker located in the genomic sequence of a FLAP gene tohybridize thereto and preferably sufficiently specific to be able todiscriminate the targeted sequence for only one nucleotide variation.

The length of these probes can range from 10, 15, 20, or 30 to 100nucleotides, preferably from 10 to 50, more preferably from 40 to 50nucleotides. A particularly preferred probe is 25 nucleotides in length.Another preferred probe is 47 nucleotides in length. It includes acentral nucleotide complementary to a polymorphic site of the FLAP gene,preferably a polymorphic site corresponding to one of the biallelicmarkers of the present invention, and a 23 nucleotide sequence spanningon each side of the central nucleotide and substantially complementaryto the nucleotide sequences of the FLAP gene spanning on each side ofthe polymorphic site. Optionally, the biallelic markers of the presentinvention comprise the polymorphic bases in the sequences of P1 to P28and the complementary sequences thereto. Optionally, the biallelicmarkers of the present invention comprise the polymorphic bases in thesequences of P1 to P13, P15, and P17 to P28, and the complementarysequences thereto.

Polymorphisms can be analyzed and the frequency of corresponding allelesquantified through hybridization reactions on amplified FLAP encodingsequences. The amplification reaction can be carried out as describedpreviously. The hybridization probes which can be conveniently used insuch reactions preferably include the probes defined above as beingsufficiently complementary to a polymorphic site defined by one of thebiallelic markers located in the genomic sequence of a FLAP gene tohybridize thereto and sufficiently specific to be able to discriminatebetween the targeted allele and an allele differing by only one base.

The target DNA comprising a biallelic marker of the present inventionmay be amplified prior to the hybridization reaction. The presence of aspecific allele in the sample is determined by detecting the presence orthe absence of stable hybrid duplexes formed between the probe and thetarget DNA. The detection of hybrid duplexes can be carried out by anumber of methods. Various detection assay formats are well known whichutilize detectable labels bound to either the target or the probe toenable detection of the hybrid duplexes. Typically, hybridizationduplexes are separated from unhybridized nucleic acids and the labelsbound to the duplexes are then detected. Those skilled in the art willrecognize that wash steps may be employed to wash away excess target DNAor probe as well as unbound conjugate. Further, standard heterogeneousassay formats are suitable for detecting the hybrids using the labelspresent on the primers and probes.

Two recently developed assays allow hybridization-based allelediscrimination with no need for separations or washes (see Landegren U.et al., 1998). The TaqMan assay takes advantage of the 5′ nucleaseactivity of Taq DNA polymerase to digest a DNA probe annealedspecifically to the accumulating amplification product. TaqMan probesare labeled with a donor-acceptor dye pair that interacts viafluorescence energy transfer. Cleavage of the TaqMan probe by theadvancing polymerase during amplification dissociates the donor dye fromthe quenching acceptor dye, greatly increasing the donor fluorescence.All reagents necessary to detect two allelic variants can be assembledat the beginning of the reaction and the results are monitored in realtime (see Livak et al., 1995). In an alternative homogeneoushybridization based procedure, molecular beacons are used for allelediscriminations. Molecular beacons are hairpin-shaped oligonucleotideprobes that report the presence of specific nucleic acids in homogeneoussolutions. When they bind to their targets they undergo a conformationalreorganization that restores the fluorescence of an internally quenchedfluorophore (Tyagi et al., 1998).

5) Hybridization to Addressable Arrays of Oligonucleotides

Efficient access to polymorphism information is obtained through a basicstructure comprising high-density arrays of oligonucleotide probesattached to a solid support (the chip) at selected positions. Each DNAchip can contain thousands to millions of individual synthetic DNAprobes arranged in a grid-like pattern and miniaturized to the size of adime. These DNA chips are detailed in “oligonucleotides primers andprobes”, section “Oligonucleotide array”.

The chip technology has already been applied with success in numerouscases. For example, the screening of mutations has been undertaken inthe BRCA1 gene, in S. cerevisiae mutant strains, and in the proteasegene of HIV-1 virus (see Hacia et al., 1996; Shoemaker et al., 1996;Kozal et al., 1996, incorporated herein by reference).

At least, three companies propose chips able to detect biallelicpolymorphisms: Affymetrix (GeneChip), Hyseq (HyChip and HyGnostics), andProtogene Laboratories.

One of the limitations encountered when using DNA chip technology isthat hybridization of nucleic acids with the probes attached to the chipin arrays is not simply a solution-phase reaction. A possibleimprovement consists in using polyacrylamide gel pads isolated from oneanother by hydrophobic regions in which the DNA probes are covalentlylinked to an acrylamide matrix.

For the detection of polymorphisms, probes which contain at least aportion of one of the biallelic markers of the present invention, suchas the biallelic markers of P1 to P28, optionally P1 to P13, P15, andP17 to P28, and the complementary sequences thereto, are synthesizedeither in situ or by conventional synthesis and immobilized on anappropriate chip using methods known to the skilled technician. Thesolid surface of the chip is often made of silicon or glass but it canbe a polymeric membrane. Thus, in some embodiments, the chips maycomprise an array of nucleic acid sequences or fragments thereof atleast 15 nucleotides in length, preferably at least 20 nucleotides inlength, and more preferably at least 25 nucleotides in length. Infurther embodiments, the chip may comprise an array including at leastone of the sequences selected from the group consisting of P1 to P28, D1to D28, and E1 to E28, or the sequences complementary thereto, or afragment thereof at least 15 consecutive nucleotides. Optionally, thechip may comprise an array including at least one of the sequencesselected from the group consisting of P1 to P13, P15, P17 to P28, D1 toD13, D15, D17 to D28, E1 to E13, E15, and E17 to E28, or the sequencescomplementary thereto, or a fragment thereof at least 15 consecutivenucleotides. In some embodiments, the chip may comprise an array of atleast 2, 3, 4, 5, 6, 7, 8 or more sequences selected from the groupconsisting of P1 to P28, D1 to D28, and E1 to E28, or the sequencescomplementary thereto, or a fragment thereof at least 15 consecutivenucleotides. Optionally, the chip may comprise an array of at least 2,3, 4, 5, 6, 7, 8 or more sequences selected from the group consisting ofP1 to P13, P15, P17 to P28, D1 to D13, D15, D17 to D28, E1 to E13, E15,and E17 to E28, or the sequences complementary thereto, or a fragmentthereof at least 15 consecutive nucleotides.

The nucleic acid sample which includes the candidate region to beanalyzed is isolated, amplified and labeled with a reporter group. Thisreporter group can be a fluorescent group such as phycoerythrin. Thelabeled nucleic acid is then incubated with the probes immobilized onthe chip using a fluidics station. For example, Manz et al. (1993)describe the fabrication of fluidics devices and particularlymicrocapillary devices, in silicon and glass substrates.

After the reaction is completed, the chip is inserted into a scanner andpatterns of hybridization are detected. The hybridization data iscollected, as a signal emitted from the reporter groups alreadyincorporated into the nucleic acid, which is now bound to the probesattached to the chip. Probes that perfectly match a sequence of thenucleic acid sample generally produce stronger signals than those thathave mismatches. Since the sequence and position of each probeimmobilized on the chip is known, the identity of the nucleic acidhybridized to a given probe can be determined.

For single-nucleotide polymorphism analyses, sets of fouroligonucleotide probes (one for each base type), preferably sets of twooligonucleotide probes (one for each base type of the biallelic marker)are generally designed that span each position of a portion of thecandidate region found in the nucleic acid sample, differing only in theidentity of the polymorphic base. The relative intensity ofhybridization to each series of probes at a particular location allowsthe identification of the base corresponding to the polymorphic base ofthe probe. Since biallelic polymorphism detection involves identifyingsingle-base mismatches on the nucleic acid sample, greater hybridizationstringencies are required (at lower salt concentration and highertemperature over shorter time periods).

The use of direct electric field control improves the determination ofsingle base mutations (Nanogen). A positive field increases thetransport rate of negatively charged nucleic acids and results in a10-fold increase of the hybridization rates. Using this technique,single base pair mismatches are detected in less than 15 sec (seeSosnowski et al., 1997).

5) Integrated Systems

Another technique, which may be used to analyze polymorphisms, includesmulticomponent integrated systems, which miniaturize andcompartmentalize processes such as PCR and capillary electrophoresisreactions in a single functional device. An example of such technique isdisclosed in U.S. Pat. No. 5,589,136, which describes the integration ofPCR amplification and capillary electrophoresis in chips.

Integrated systems can be envisaged mainly when microfluidic systems areused. These systems comprise a pattern of microchannels designed onto aglass, silicon, quartz, or plastic wafer included on a microchip. Themovements of the samples are controlled by electric, electroosmotic orhydrostatic forces applied across different areas of the microchip tocreate functional microscopic valves and pumps with no moving parts.Varying the voltage controls the liquid flow at intersections betweenthe micro-machined channels and changes the liquid flow rate for pumpingacross different sections of the microchip.

For genotyping biallelic markers, the microfluidic system may integratenucleic acid amplification, microsequencing, capillary electrophoresisand a detection method such as laser-induced fluorescence detection. Ina first step, the DNA samples are amplified, preferably by PCR. Then,the amplification products are subjected to automated microsequencingreactions using ddNTPs (specific fluorescence for each ddNTP) and theappropriate oligonucleotide microsequencing primers which hybridize justupstream of the targeted polymorphic base. Once the extension at the 3′end is completed, the primers are separated from the unincorporatedfluorescent ddNTPs by capillary electrophoresis. The separation mediumused in capillary electrophoresis can for example be polyacrylamide,polyethyleneglycol or dextran. The incorporated ddNTPs in thesingle-nucleotide primer extension products are identified byfluorescence detection. This microchip can be used to process at least96 to 384 samples in parallel. It can use the usual four color laserinduced fluorescence detection of the ddNTPs.

IX. Association Studies

The identification of genes associated with a particular trait such asasthma susceptibility or individual response to anti-asthmatic drugs canbe carried out through two main strategies currently used for geneticmapping: linkage analysis and association studies. Linkage analysisinvolves the study of families with multiple affected individuals and isnow useful in the detection of mono- or oligogenic inherited-traits.Conversely, association studies examine the frequency of marker allelesin unrelated trait positive (T+) individuals compared with controlindividuals who are randomly selected or preferably trait negative (T−)controls, and are generally employed in the detection of polygenicinheritance.

Association studies as a method of mapping genetic traits rely on thephenomenon of linkage disequilibrium. If two genetic loci lie on thesame chromosome, then sets of alleles of these loci on the samechromosomal segment (called haplotypes) tend to be transmitted as ablock from generation to generation. When not broken up byrecombination, haplotypes can be tracked not only through pedigrees butalso through populations. The resulting phenomenon at the populationlevel is that the occurrence of pairs of specific alleles at differentloci on the same chromosome is not random, and the deviation from randomis called linkage disequilibrium (LD).

If a specific allele in a given gene is directly involved in causing aparticular trait T, its frequency will be statistically increased in aT+ population when compared to the frequency in a T− population. As aconsequence of the existence of linkage disequilibrium, the frequency ofall other alleles present in the haplotype carrying the trait-causingallele (TCA) will also be increased in T+ individuals compared to T−individuals. Therefore, association between the trait and any allele inlinkage disequilibrium with the trait-causing allele will suffice tosuggest the presence of a trait-related gene in that particular allele'sregion. Linkage disequilibrium allows the relative frequencies in T+ andT− populations of a limited number of genetic polymorphisms(specifically biallelic markers) to be analyzed as an alternative toscreening all possible functional polymorphisms in order to findtrait-causing alleles.

Two alternative approaches can be employed to perform associationstudies: a genome-wide association study and a candidate geneassociation study. The genome-wide association study relies on thescreening of genetic markers evenly spaced and covering the entiregenome. The candidate gene approach is based on the study of geneticmarkers specifically located in genes potentially involved in abiological pathway related to the trait of interest. The candidate geneanalysis clearly provides a short-cut approach to the identification ofgenes and gene polymorphisms related to a particular trait when someinformation concerning the biology of the trait is available.

The general strategy to perform association studies using biallelicmarkers derived from a candidate gene is to scan two group ofindividuals (trait+ and trait− control individuals which arecharacterized by a well defined phenotype as described below) in orderto measure and statistically compare the allele frequencies of suchbiallelic markers in both groups.

If a statistically significant association with a trait is identifiedfor at least one or more of the analyzed biallelic markers, one canassume that: either the associated allele is directly responsible forcausing the trait (the associated allele is the TCA), or the associatedallele is in linkage disequilibrium with the TCA. The specificcharacteristics of the associated allele with respect to the candidategene function usually gives further insight into the relationshipbetween the associated allele and the trait (causal or in linkagedisequilibrium). If the evidence indicates that the associated allelewithin the candidate gene is most probably not the TCA but is in linkagedisequilibrium with the real TCA, then the TCA can be found bysequencing the vicinity of the associated marker.

It is another object of the present invention to provide a method forthe identification and characterization of an association betweenalleles for one or several biallelic markers of the sequence of the FLAPgene and a trait. The method of detecting an association between agenotype and a trait, comprising the steps of: a) determining thefrequency of at least one FLAP-related biallelic marker in traitpositive population according to a method of the invention; b)determining the frequency of at least one FLAP-related biallelic markerin a control population according to a method of the invention; and c)determining whether a statistically significant association existsbetween said genotype and said trait; Optionally, said biallelic markersare selected from the group consisting of A1 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; Optionally, said FLAP-related biallelic marker may beselected from the group consisting of A1 to A13, A15, and A17 to A28,and the complements thereof, or optionally the biallelic markers inlinkage disequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A1 to A10 and A22 to A28, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A11 to A13, A15, A17 to A21, andthe complements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A14 or A16, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith. Optionally, the trait is either a disease, preferably adisease involving the leukotriene pathway, most preferably asthma, abeneficial response to treatment with agents acting on the leukotrienepathway or side-effects related to treatment with agents acting on theleukotriene pathway. Optionally, said genotyping steps a) and b) may beperformed on a pooled biological sample derived from each of saidpopulations; Optionally, said genotyping steps a) and b) are performedseparately on biological samples derived from each individual in saidpopulation or a subsample thereof; Optionally, said control individualsare trait negative or random controls.

The invention also encompasses methods of estimating the frequency of ahaplotype for a set of biallelic markers in a population, comprising thesteps of: a) genotyping at least one FLAP-related biallelic markeraccording to a method of the invention for each individual in saidpopulation; b) genotyping a second biallelic marker by determining theidentity of the nucleotides at said second biallelic marker for bothcopies of said second biallelic marker present in the genome of eachindividual in said population; and c) applying a haplotype determinationmethod to the identities of the nucleotides determined in steps a) andb) to obtain an estimate of said frequency. In addition, the methods ofestimating the frequency of a haplotype of the invention encompassmethods with any further limitation described in this disclosure,particularly in “Statistical methods”, or those following, specifiedalone or in any combination; Optionally, said biallelic markers areselected from the group consisting of A1 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; Optionally, said FLAP-related biallelic marker may beselected from the group consisting of A1 to A13. A15, and A17 to A28,and the complements thereof, or optionally the biallelic markers inlinkage disequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A1 to A10 and A22 to A28, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A11 to A13, A15, A17 to A21, andthe complements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith; optionally, said biallelic markers areselected from the group consisting of A14 or A16, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith. Optionally, said haplotype determination method is performedby asymmetric PCR amplification, double PCR amplification of specificalleles, the Clark algorithm, or an expectation-maximization algorithm.

The present invention also provides a method for the identification andcharacterization of an association between a haplotype comprisingalleles for several biallelic markers of the genomic sequence of theFLAP gene and a trait. The method comprises the steps of: a) genotypinga group of biallelic markers according to the invention in traitpositive and control individuals; and b) establishing a statisticallysignificant association between a haplotype and the trait. In a furtherembodiment, a method for the identification and characterization of anassociation between a haplotype comprising alleles for several biallelicmarkers of the genomic sequence of the FLAP gene and a trait comprisesthe steps of: a) estimating the frequency of at least one haplotype in atrait positive population according to a method of the invention; b)estimating the frequency of said haplotype in a control populationaccording to a method of the invention; and c) determining whether astatistically significant association exists between said haplotype andsaid trait. In addition, the methods of detecting an association betweena haplotype and a phenotype of the invention encompass methods with anyfurther limitation described in this disclosure, or those following;Optionally, said biallelic markers are selected from the groupconsisting of A1 to A28, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith; Optionally, saidFLAP-related biallelic marker may be selected from the group consistingof A1 to A13, A15, and A17 to A28, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith;optionally, said biallelic markers are selected from the groupconsisting of A1 to A10 and A22 to A28, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith;optionally, said biallelic markers are selected from the groupconsisting of A11 to A13, A15, A17 to A21, and the complements thereof,or optionally the biallelic markers in linkage disequilibrium therewith;optionally, said biallelic markers are selected from the groupconsisting of A14 or A16, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith. Optionally, thetrait is a disease, preferably a disease involving the leukotrienepathway, most preferably asthma, a beneficial response to treatment withagents acting on the leukotriene pathway or side-effects related totreatment with agents acting on the leukotriene pathway; Optionally,said control individuals are trait negative or random controls.Optionally, said method comprises the additional steps of determiningthe phenotype in said trait positive and said control populations priorto step c).

If the trait is a beneficial response or conversely a side-effect totreatment with an agent acting on the leukotriene pathway, the method ofthe invention referred to above further comprises some or all of thefollowing steps: a) selecting a population or cohort of subjectsdiagnosed as suffering from a specified disease involving theleukotriene pathway; b) administering a specified agent acting on theleukotriene pathway to said cohort of subjects; c) monitoring theoutcome of drug administration and identifying those individuals thatare trait positive or trait negative relative to the treatment; d)taking from said cohort biological samples containing DNA and testingthis DNA for the presence of a specific allele or of a set of allelesfor biallelic markers of the FLAP gene; e) analyzing the distribution ofalleles for biallelic markers between trait positive and trait negativeindividuals; and, f) performing a statistical analysis to determine ifthere is a statistically significant association between the presence orabsence of alleles of biallelic markers of the FLAP gene and thetreatment related trait. Optionally, said biallelic markers are selectedfrom the group consisting of A1 to A28, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith;Optionally, said FLAP-related biallelic marker may be selected from thegroup consisting of A1 to A13, A15, and A17 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, said biallelic markers are selected from thegroup consisting of A1 to A10 and A22 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, said biallelic markers are selected from thegroup consisting of A11 to A13, A15, A17 to A21, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, said biallelic markers are selected from thegroup consisting of A14 or A16 and the complements thereof. The step oftesting for and detecting the presence of DNA comprising specificalleles of a biallelic marker or a group of biallelic markers of thepresent invention can be carried out as described in the presentinvention.

The invention also encompasses methods of determining whether anindividual is at risk of developing asthma, comprising the steps of: a)genotyping at least one FLAP-related biallelic marker according to amethod of the present invention; and b) correlating the result of stepa) with a risk of developing asthma; optionally wherein saidFLAP-related biallelic marker is selected from the group consisting ofA1 to A28; optionally, wherein said FLAP-related biallelic marker isselected from the following list of biallelic markers: A2, A14, A16,A18, A19, A22, and A23; and optionally, wherein said FLAP-relatedbiallelic marker is the biallelic marker A19.

1) Collection of DNA Samples from Trait Positive (Trait+) and ControlIndividuals (Inclusion Criteria)

Population-based association studies do not concern familial inheritancebut compare the prevalence of a particular genetic marker, or a set ofmarkers, in case-control populations. They are case-control studiesbased on comparison of unrelated case (affected or trait positive)individuals and unrelated control (unaffected or trait negative orrandom) individuals. Preferably the control group is composed ofunaffected or trait negative individuals. Further, the control group isethnically matched to the case population. Moreover, the control groupis preferably matched to the case-population for the main knownconfusion factor for the trait under study (for example age-matched foran age-dependent trait). Ideally, individuals in the two samples arepaired in such a way that they are expected to differ only in theirdisease status. In the following “trait positive population”, “casepopulation” and “affected population” are used interchangeably.

In order to perform efficient and significant association studies suchas those described herein, the trait under study should preferablyfollow a bimodal distribution in the population under study, presentingtwo clear non-overlapping phenotypes, trait+ and trait−.

Nevertheless, even in the absence of such bimodal distribution (as mayin fact be the case for more complex genetic traits), any genetic traitmay still be analyzed by the association method proposed here bycarefully selecting the individuals to be included in the trait+ andtrait− phenotypic groups. The selection procedure involves selectingindividuals at opposite ends of the non-bimodal phenotype spectra of thetrait under study, so as to include in these trait+ and trait−populations individuals which clearly represent extreme, preferablynon-overlapping phenotypes.

The definition of the inclusion criteria for the trait+ and trait−populations is an important aspect of the present invention.

Typical examples of inclusion criteria include a disease involving theleukotriene pathway such as asthma or the evaluation of livertransaminase levels following treatment with an anti-asthma drug such asZileuton. From a statistical viewpoint, if one considers that in a givenpopulation liver transaminase levels follow a standard distributioncurve, individuals with extreme phenotypes according to the optimalinclusion criteria would correspond respectively to those exhibiting thelowest liver transaminase levels and those exhibiting the highest livertransaminase levels.

The selection of those drastically different but relatively uniformphenotypes enables efficient comparisons in association studies and thepossible detection of marked differences at the genetic level, providedthat the sample sizes of the populations under study are significantenough.

Generally, trait+ and trait− populations to be included in associationstudies such as those described in the present application consist ofphenotypically homogenous populations of individuals each representing100% of the corresponding trait if the trait distribution is bimodal.

If the trait distribution is non-bimodal, trait+ and trait− populationsconsist of phenotypically uniform populations of individualsrepresenting between 1 and 98%, preferably between 1 and 80%, morepreferably between 1 and 50%, and most preferably between 4 and 35% ofthe total population under study, and selected from individualsexhibiting the extreme phenotypes of the group. The clearer is thedifference between the two trait phenotypes, the greater is theprobability to observe an association with biallelic markers.

A first group of between 50 and 300 trait+ individuals, preferably about100 individuals, are recruited according to clinical inclusion criteriabased on either 1) affection by disease(s) involving the leukotrienepathway, preferably asthma, 2) evidence of side-effects observedfollowing administration of an agent acting on the leukotriene pathway,preferably increased liver transaminase levels following administrationof Zileuton, or 3) evidence of particular responses to treatment withagents acting on the leukotriene pathway.

In each case, a similar number of trait negative individuals areincluded in such studies. They are checked for the absence of theclinical criteria defined above. Both trait+ and trait− individualsshould be unrelated cases.

In the context of the present invention, one association study werecarried out. The considered trait was asthma. Collection of DNA samplesfrom trait+ and trait− individuals is described in Example 5.

2) Genotyping of Trait+ and Trait− Individuals

Allelic frequencies of the biallelic markers in each of the abovedescribed populations can be determined using one of the methodsdescribed above under the heading “Methods of Genotyping DNA samples forBiallelic Markers”. Analyses are preferably performed on amplifiedfragments obtained by genomic PCR performed on the DNA samples from eachindividual in similar conditions as those described above for thegeneration of biallelic markers.

In a preferred embodiment, amplified DNA samples are subjected toautomated microsequencing reactions using fluorescent ddNTPs (specificfluorescence for each ddNTP) and the appropriate oligonucleotidemicrosequencing primers which hybridize just upstream of the polymorphicbase. Genotyping is further described in Example 5.

3) Single Marker Association Studies and Haplotype Frequency Analysis

Methods for determining the statistical significance of a correlationbetween a phenotype and a genotype, in this case an allele at abiallelic marker or a haplotype made up of such alleles, may bedetermined by any statistical test known in the art and with anyaccepted threshold of statistical significance being required. Theapplication of particular methods and thresholds of significance arewell with in the skill of the ordinary practitioner of the art.

Testing for association is performed by determining the frequency of abiallelic marker allele in case and control populations and comparingthese frequencies with a statistical test to determine if there is astatistically significant difference in frequency which would indicate acorrelation between the trait and the biallelic marker allele understudy. Similarly, a haplotype analysis is performed by estimating thefrequencies of all possible haplotypes for a given set of biallelicmarkers in case and control populations, and comparing these frequencieswith a statistical test to determine if their is a statisticallysignificant correlation between the haplotype and the phenotype (trait)under study. Any statistical tool useful to test for a statisticallysignificant association between a genotype and a phenotype may be used.Preferably the statistical test employed is a chi-square test with onedegree of freedom. A P-value is calculated (the P-value is theprobability that a statistic as large or larger than the observed onewould occur by chance).

In preferred embodiments, significance for diagnosis purposes, either asa positive basis for further diagnostic tests or as a preliminarystarting point for early preventive therapy, the p value related to abiallelic marker association is preferably about 1×10⁻² or less, morepreferably about 1×10⁻⁴ or less, for a single biallelic marker analysisand about 1×10⁻³ or less, still more preferably 1×10⁻⁶ or less and mostpreferably of about 1×10⁻⁸ or less, for a haplotype analysis involvingtwo or more markers. These values are believed to be applicable to anyassociation studies involving single or multiple marker combinations.

The skilled person can use the range of values set forth above as astarting point in order to carry out association studies with biallelicmarkers of the present invention. In doing so, significant associationsbetween the biallelic markers of the present invention and a diseaseinvolving the leukotriene pathway can be revealed and used for diagnosisand drug screening purposes.

To address the problem of false positives similar analysis may beperformed with the same case-control populations in random genomicregions. Results in random regions and the candidate region are comparedas described in a co-pending US Provisional patent application entitled“Methods, Software And Apparati For Identifying Genomic RegionsHarboring A Gene Associated With A Detectable Trait,” U.S. Ser. No.60/107,986, filed Nov. 10, 1998, the contents of which are incorporatedherein by reference.

Single Marker Association

Association studies are usually run in two successive steps. In a firstphase, the frequencies of a reduced number of biallelic markers, usuallybetween 2 and 10 markers, is determined in the trait+ and trait−populations. In a second phase of the analysis, the position of thegenetic loci responsible for the given trait is further refined using ahigher density set of markers. However, if the candidate gene understudy is relatively small in length, as it is the case for the FLAPgene, it is believed that a single phase is sufficient to establishsignificant associations.

In one preferred embodiment of the invention in which a correlation wasfound between a set of biallelic markers of the FLAP gene and a diseaseinvolving the leukotriene pathway, more particularly asthma, results ofthe first step of the association study, further details of which areprovided in example 7, seem to indicate that asthma is associated moststrongly with the biallelic marker A19 (10-35/390, allele T). Furtherdetails concerning these associations are provided in Example 7.

Similar association studies can also be carried out with other biallelicmarkers within the scope of the invention, preferably with biallelicmarkers in linkage disequilibrium with the markers associated withasthma, including the biallelic markers A1 to A28.

Similar associations studies can be routinely carried out by the skilledtechnician using the biallelic markers of the invention which aredefined above with different trait+ and trait− populations. Suitablefurther examples of possible association studies using biallelic markersof the FLAP gene, including the biallelic markers A1 to A28, involvestudies on the following populations:

-   -   a trait+ population suffering from a disease involving the        leukotriene pathway and a healthy unaffected population; or    -   a trait+ population treated with agents acting on the        leukotriene pathway suffering from side-effects resulting from        the treatment and an trait− population treated with same agents        without any side-effects; or    -   a trait+ population treated with agents acting on the        leukotriene pathway showing a beneficial response and a trait−        population treated with same agents without any beneficial        response.

Haplotype Frequency Analysis

A haplotype analysis is interesting in that it increases the statisticalsignificance of an analysis involving individual markers. Indeed, bycombining the informativeness of a set of biallelic markers, itincreases the value of the results obtained through associationanalyses, allowing false positive and/or negative data that may resultfrom the single marker studies to be eliminated.

In a first stage of a haplotype frequency analysis, the frequency of thepossible haplotypes based on various combinations of the identifiedbiallelic markers of the invention is determined and compared fordistinct populations of trait+ and trait− individuals. The number oftrait+ individuals which should be subjected to this analysis to obtainstatistically significant results usually ranges between 30 and 300,with a preferred number of individuals ranging between 50 and 150. Thesame considerations apply to the number of unaffected controls used inthe study.

The results of this first analysis provide haplotype frequencies for thetested trait+ and trait− individuals, and the estimated p value for eachevaluated haplotype.

In the association of the biallelic markers of FLAP gene with theasthma, several haplotypes were also shown to be significant (see FIG.3). For example, the preferred haplotypes comprise the allele T of thebiallelic marker A19 (10-35/390). The more preferred haplotype (HAP 1 ofFIG. 3) comprise the allele A of the marker A14 (10-33/234) and theallele T of the marker A19 (10-35/390). This haplotype is considered tobe highly significant of an association with asthma. The othersignificant haplotypes are detailed in Example 8.

In order to confirm the statistical significance of the first stagehaplotype analysis described above, it might be suitable to performfurther analyses in which genotyping data from case-control individualsare pooled and randomized with respect to the trait phenotype. Eachindividual genotyping data is randomly allocated to two groups, whichcontain the same number of individuals as the case-control populationsused to compile the data obtained in the first stage. A second stagehaplotype analysis is preferably run on these artificial groups,preferably for the markers included in the haplotype of the first stageanalysis showing the highest relative risk coefficient. This experimentis reiterated preferably at least between 100 and 10000 times. Therepeated iterations allow the determination of the probability to obtainby chance the tested haplotype.

For the association between asthma and the three considered haplotypes,a randomized haplotype analysis was reiterated 1000 times or 10000 timesand the results are shown in FIG. 4. These results demonstrate thatamong 1000 iterations none and among 10,000 iterations only 1 of theobtained haplotypes had a p-value comparable to the one obtained for thehaplotype HAP1. These results clearly validate the statisticalsignificance of the association between this haplotype and asthma.

Using the method described above and evaluating the associations forsingle marker alleles or for haplotypes permits an estimation of therisk a corresponding carrier has to develop a given trait, andparticularly in the context of the present invention, a disease,preferably a disease involving the leukotriene pathway, more preferablyasthma. Significance thresholds of relative risks are to be adapted tothe reference sample population used. The evaluation of the risk factorsis detailed in “Statistical methods”.

It will of course be understood by practitioners skilled in thetreatment of diseases involving the leukotriene pathway listed above,and in particular asthma, that the present invention does not intend toprovide an absolute identification of individuals who could be at riskof developing a particular disease involving the leukotriene pathway orwho will or will not respond or exhibit side-effects to treatment withagents acting on the leukotriene pathway but rather to indicate acertain degree or likelihood of developing a disease or of observing ina given individual a response or a side-effect to treatment with saidagents.

However, this information is extremely valuable as it can, in certaincircumstances, be used to initiate preventive treatments or to allow anindividual carrying a significant haplotype to foresee warning signssuch as minor symptoms. In diseases such as asthma, in which attacks maybe extremely violent and sometimes fatal if not treated on time, theknowledge of a potential predisposition, even if this predisposition isnot absolute, might contribute in a very significant manner to treatmentefficacy. Similarly, a diagnosed predisposition to a potentialside-effect could immediately direct the physician toward a treatmentfor which such side-effects have not been observed during clinicaltrials.

X. Statistical Methods

In general, any method known in the art to test whether a trait and agenotype show a statistically significant correlation may be used.

1) Methods to Estimate Haplotype Frequencies in a Population

The gametic phase of haplotypes is unknown when diploid individuals areheterozygous at more than one locus. Using genealogical information infamilies gametic phase can sometimes be inferred (Perlin et al., 1994).When no genealogical information is available different strategies maybe used. One possibility is that the multiple-site heterozygous diploidscan be eliminated from the analysis, keeping only the homozygotes andthe single-site heterozygote individuals, but this approach might leadto a possible bias in the sample composition and the underestimation oflow-frequency haplotypes. Another possibility is that single chromosomescan be studied independently, for example, by asymmetric PCRamplification (see Newton et al, 1989; Wu et al., 1989) or by isolationof single chromosomes by limit dilution followed by PCR amplification(see Ruano et al., 1990). Further, a sample may be haplotyped forsufficiently close biallelic markers by double PCR amplification ofspecific alleles (Sarkar, G. and Sommer S. S., 1991). These approachesare not entirely satisfying either because of their technicalcomplexity, the additional cost they entail, their lack ofgeneralization at a large scale, or the possible biases they introduce.To overcome these difficulties, an algorithm to infer the phase ofPCR-amplified DNA genotypes introduced by Clark, A. G. (1990) may beused. Briefly, the principle is to start filling a preliminary list ofhaplotypes present in the sample by examining unambiguous individuals,that is, the complete homozygotes and the single-site heterozygotes.Then other individuals in the same sample are screened for the possibleoccurrence of previously recognized haplotypes. For each positiveidentification, the complementary haplotype is added to the list ofrecognized haplotypes, until the phase information for all individualsis either resolved or identified as unresolved. This method assigns asingle haplotype to each multiheterozygous individual, whereas severalhaplotypes are possible when there are more than one heterozygous site.Alternatively, one can use methods estimating haplotype frequencies in apopulation without assigning haplotypes to each individual. Preferably,a method based on an expectation-maximization (EM) algorithm (Dempsteret al., 1977) leading to maximum-likelihood estimates of haplotypefrequencies under the assumption of Hardy-Weinberg proportions (randommating) is used (see Excoffier L. and Slatkin M., 1995). The EMalgorithm is a generalized iterative maximum-likelihood approach toestimation that is useful when data are ambiguous and/or incomplete. TheEM algorithm is used to resolve heterozygotes into haplotypes. The EMalgorithm can be applied using for example the EM-HAPLO program (HawleyM. E. et al., 1994) or the Arlequin program (Schneider et al., 1997).Any other method known in the art to determine or to estimate thefrequency of a haplotype in a population may be used (see Lange K.,1997; Weir, B. S., 1996). The EM algorithm is briefly described below.

A sample of N unrelated individuals is typed for K markers. The dataobserved are the unknown-phase K-locus phenotypes that can categorizedin F different phenotypes. Suppose that we have H underlying possiblehaplotypes (in case of K biallelic markers, H=2^(K)).

For phenotype j, suppose that c_(j) genotypes are possible. We thus havethe following equation

$\begin{matrix}{P_{j} = {{\sum\limits_{i = 1}^{c_{j}}{{pr}( {genotype}_{i} )}} = {\sum\limits_{i = 1}^{c_{j}}{{pr}( {h_{k},h_{l}} )}}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

where Pj is the probability of the phenotype j, h_(k) and h_(l) are thetwo haplotypes constituent the genotype i. Under the Hardy-Weinbergequilibrium, pr(h_(k),h_(l)) becomes:pr(h _(k) ,h _(l))=pr(h _(k))² if h _(k) =h _(l) ,pr(h _(k) ,h_(l))=2pr(h _(k))·pr(h _(l)) if h _(k) ≠h _(l).  Equation 2

The successive steps of the E-M algorithm can be described as follows:

Starting with initial values of the of haplotypes frequencies, noted p₁⁽⁰⁾, p₂ ⁽⁰⁾, . . . , p_(H) ⁽⁰⁾, these initial values serve to estimatethe genotype frequencies (Expectation step) and then estimate anotherset of haplotype frequencies (Maximization step), noted p₁ ⁽¹⁾, p₂ ⁽¹⁾,. . . p_(H) ⁽¹⁾ these two steps are iterated until changes in the setsof haplotypes frequency are very small.

A stop criterion can be that the maximum difference between haplotypefrequencies between two iterations is less than 10⁻⁷. These values canbe adjusted according to the desired precision of estimations.

At a given iteration s, the Expectation step consists in calculating thegenotypes frequencies by the following equation:

$\begin{matrix}\begin{matrix}{{{pr}( {genotype}_{i} )}^{(s)} = {{{pr}( {phenotype}_{j} )} \cdot}} \\{{{pr}( {genotype}_{i} \middle| {phenotype}_{j} )}^{(s)}} \\{= {\frac{n_{j}}{N} \cdot \frac{{{pr}( {h_{k},h_{l}} )}^{(s)}}{P_{j}^{(s)}}}}\end{matrix} & {{Equation}\mspace{14mu} 3}\end{matrix}$

where genotype i occurs in phenotype j, and where h_(k) and h_(l)constitute genotype i. Each probability is derived according to eq. 1,and eq. 2 described above.

Then the Maximization step simply estimates another set of haplotypefrequencies given the genotypes frequencies. This approach is also knownas the gene-counting method (Smith, 1957).

$\begin{matrix}{p_{t}^{({s + 1})} = {\frac{1}{2}{\sum\limits_{j = 1}^{F}{\sum\limits_{i = 1}^{c_{j}}{\delta_{it} \cdot {{pr}( {genotype}_{i} )}^{(s)}}}}}} & {{Equation}\mspace{14mu} 4}\end{matrix}$

Where δ_(it) is an indicator variable which count the number of timehaplotype t in genotype i. It takes the values of 0, 1 or 2.

To ensure that the estimation finally obtained is the maximum-likelihoodestimation several values of departures are required. The estimationsobtained are compared and if they are different the estimations leadingto the best likelihood are kept.

2) Methods to Calculate Linkage Disequilibrium Between Markers

A number of methods can be used to calculate linkage disequilibriumbetween any two genetic positions, in practice linkage disequilibrium ismeasured by applying a statistical association test to haplotype datataken from a population.

Linkage disequilibrium between any pair of biallelic markers comprisingat least one of the biallelic markers of the present invention (M_(i),M_(j)) having alleles (a_(i)/b_(i)) at marker M_(i) and alleles(a_(j)/b_(j)) at marker M_(j) can be calculated for every allelecombination (a_(i),a_(j); a_(i),b_(j); b_(i),a_(j) and b_(i),b_(j)),according to the Piazza formula:Δ_(aiaj)=√θ4−√(θ4+θ3)(θ4+θ2), where:

θ4=−−=frequency of genotypes not having allele a_(i) at M_(i) and nothaving allele a_(i) at M_(j)

θ3=−+=frequency of genotypes not having allele a_(i) at M_(i) and havingallele a_(j) at M_(i)

θ2=+−=frequency of genotypes having allele a_(i) at M_(i) and not havingallele a_(j) at M_(j)

Linkage disequilibrium (LD) between pairs of biallelic markers (M_(i),M_(j)) can also be calculated for every allele combination (ai,aj;ai,bj, b_(i),a_(j) and b_(i),b_(j)), according to the maximum-likelihoodestimate (MLE) for delta (the composite genotypic disequilibriumcoefficient), as described by Weir (Weir B. S., 1996). The MLE for thecomposite linkage disequilibrium is:D _(aiaj)=(2n ₁ +n ₂ +n ₃ +n ₄/2)/N−2(pr(a _(i))·pr(a _(j)))

Where n₁=Σ phenotype (a_(i)/a_(i), a_(j)/a_(j)), n₂=Σ phenotype(a_(i)/a_(i), a_(i)/b_(j)), n₃=Σ phenotype (a_(i)/b_(i), a_(j)/a_(j)),n₄=Σ phenotype (a_(i)/b_(i), a_(j)/b_(j)) and N is the number ofindividuals in the sample.

This formula allows linkage disequilibrium between alleles to beestimated when only genotype, and not haplotype, data are available.

Another means of calculating the linkage disequilibrium between markersis as follows. For a couple of biallelic markers, M_(i)(a_(i)/b_(i)) andM_(j)(a_(j)/b_(j)), fitting the Hardy-Weinberg equilibrium, one canestimate the four possible haplotype frequencies in a given populationaccording to the approach described above.

The estimation of gametic disequilibrium between ai and aj is simply:D _(aiaj) =pr(haplotype(a _(i) ,a _(j)))−pr(a _(i))·pr(a _(j)).

Where pr(a_(i)) is the probability of allele a_(i) and pr(a_(j)) is theprobability of allele a_(j) and where pr(haplotype (a_(i), a_(j))) isestimated as in Equation 3 above.

For a couple of biallelic markers only one measure of disequilibrium isnecessary to describe the association between M_(i) and M_(j).

Then a normalized value of the above is calculated as follows:D′ _(aiaj) =D _(aiaj)/max(−pr(a _(i))·pr(a _(j)),−pr(b _(i))·pr(b _(j)))with D _(aiaj)<0D′ _(aiaj) =D _(aiaj)/max(pr(b _(j))·pr(a _(j)),pr(a _(i))·pr(b _(j)))with D _(aiaj)>0

The skilled person will readily appreciate that other linkagedisequilibrium calculation methods can be used.

Linkage disequilibrium among a set of biallelic markers having anadequate heterozygosity rate can be determined by genotyping between 50and 1000 unrelated individuals, preferably between 75 and 200, morepreferably around 100.

3) Evaluation Of Risk Factors

The association between a risk factor (in genetic epidemiology the riskfactor is the presence or the absence of a certain allele or haplotypeat marker loci) and a disease is measured by the odds ratio (OR) and bythe relative risk (RR). If P(R⁺) is the probability of developing thedisease for individuals with R and P(R⁻) is the probability forindividuals without the risk factor, then the relative risk is simplythe ratio of the two probabilities, that is:RR=P(R⁺)/P(R⁻)

In case-control studies, direct measures of the relative risk cannot beobtained because of the sampling design. However, the odds ratio allowsa good approximation of the relative risk for low-incidence diseases andcan be calculated:OR=(F⁺/(1−F⁻))/(F⁻/(1−F⁻))

F⁺ is the frequency of the exposure to the risk factor in cases and F⁻is the frequency of the exposure to the risk factor in controls. F⁺ andF⁻ are calculated using the allelic or haplotype frequencies of thestudy and further depend on the underlying genetic model (dominant,recessive, additive . . . ).

One can further estimate the attributable risk (AR) which describes theproportion of individuals in a population exhibiting a trait due to agiven risk factor. This measure is important in quantifying the role ofa specific factor in disease etiology and in terms of the public healthimpact of a risk factor. The public health relevance of this measurelies in estimating the proportion of cases of disease in the populationthat could be prevented if the exposure of interest were absent. AR isdetermined as follows:AR=P_(E)(RR−1)/(P_(E)(RR−1)+1)

AR is the risk attributable to a biallelic marker allele or a biallelicmarker haplotype. P_(E) is the frequency of exposure to an allele or ahaplotype within the population at large; and RR is the relative riskwhich, is approximated with the odds ratio when the trait under studyhas a relatively low incidence in the general population.

XI. Identification of Biallelic Markers in Linkage Disequilibrium withthe Biallelic Markers of the Present Invention

Once a first biallelic marker has been identified in a genomic region ofinterest, the practitioner of ordinary skill in the art, using theteachings of the present invention, can easily identify additionalbiallelic markers in linkage disequilibrium with this first marker. Asmentioned before any marker in linkage disequilibrium with a firstmarker associated with a trait will be associated with the trait.Therefore, once an association has been demonstrated between a givenbiallelic marker and a trait, the discovery of additional biallelicmarkers associated with this trait is of great interest in order toincrease the density of biallelic markers in this particular region. Thecausal gene or mutation will be found in the vicinity of the marker orset of markers showing the highest correlation with the trait.

The invention also concerns a method for the identification andcharacterization of a biallelic marker in linkage disequilibrium with abiallelic marker of a FLAP gene, preferably a biallelic marker of a FLAPgene of which one allele is associated with a trait. In one embodiment,the biallelic marker in linkage disequilibrium with a biallelic markerof the FLAP gene is in the genomic region harboring the FLAP gene, butoutside of the FLAP gene itself. In another embodiment, the biallelicmarker in linkage disequilibrium with a biallelic marker of the FLAPgene is itself located within the FLAP gene. The method comprises thefollowing steps: a) amplifying a genomic fragment comprising a firstbiallelic marker from a plurality of individuals; b) identifying secondbiallelic markers in the genomic region harboring the first biallelicmarker; c) conducting a linkage disequilibrium analysis between saidfirst biallelic marker and second biallelic markers; and d) selectingsaid second biallelic markers in linkage disequilibrium with said firstmarker.

In one embodiment, the step of sequencing and identifying secondbiallelic markers comprises sequencing second biallelic markers withinthe FLAP gene. In a further embodiment, the step of sequencing andidentifying second biallelic markers comprises sequencing secondbiallelic markers within the amplified region of the FLAP gene.

Once identified, the sequences in linkage disequilibrium with abiallelic marker of the FLAP gene may be used in any of the methodsdescribed herein, including methods for determining an associationbetween biallelic marker and a trait, methods for identifyingindividuals having a predisposition for a trait, methods of diseasetreatment, methods of identifying individuals likely to respondpositively or negatively to drug treatment, and methods of using drugs.In particular, biallelic markers in linkage disequilibrium with abiallelic marker in the FLAP gene may be used to identify individualshaving a predisposition to asthma or to positive or negative responsesto treatment with anti-asthma drugs such as Zileuton.

Methods to identify biallelic markers and to conduct linkagedisequilibrium analysis are described herein in “Statistical methods”and can be carried out by the skilled person without undueexperimentation. The present invention then also concerns biallelicmarkers which are in linkage disequilibrium with the specific biallelicmarkers A1 to A28 and which are expected to present similarcharacteristics in terms of their respective association with a giventrait.

XII. Identification of Trait-Causing Mutations in the FLAP Gene

Mutations in the FLAP gene which are responsible for a detectablephenotype may be identified by comparing the sequences of the FLAP genesfrom trait-positive and trait-negative individuals. Preferably, trait+individuals to be sequenced carry the haplotype shown to be associatedto the trait and trait− individuals to be sequenced do not carry thehaplotype associated to the trait. The detectable phenotype may comprisea variety of manifestations of altered FLAP function, including adisease involving the leukotriene pathway, a response to an agent actingon the leukotriene pathway or side-effects linked to a treatment withthis agent. The mutations may comprise point mutations, deletions, orinsertions in the FLAP gene. The mutations may lie within the codingsequence for the FLAP protein or within regulatory regions in the FLAPgene.

The method used to detect such mutations generally comprises thefollowing steps: a) amplification of a region of the FLAP genecomprising a biallelic marker or a group of biallelic markers associatedwith the trait from DNA samples of trait positive patients and traitnegative controls; b) sequencing of the amplified region; c) comparisonof DNA sequences from trait-positive patients and trait-negativecontrols; and, d) determination of mutations specific to trait-positivepatients.

Oligonucleotide primers are constructed as described previously toamplify the sequences of each of the exons, introns or the promoterregion of the FLAP gene.

Each primer pair is used to amplify the exon or promoter region fromwhich it is derived. Amplification is carried out on genomic DNA samplesfrom trait positive patients and trait negative controls, preferablyusing the PCR conditions described in the examples. Amplificationproducts from the genomic PCRs are then subjected to sequencing,preferably through automated dideoxy terminator sequencing reactions andelectrophoresed, preferably on ABI 377 sequencers. Following gel imageanalysis and DNA sequence extraction, ABI sequence data areautomatically analyzed to detect the presence of sequence variationsamong trait positive and trait negative individuals. Sequences areverified by determining the sequences of both DNA strands for eachindividual.

Candidate polymorphisms suspected of being responsible for thedetectable phenotype, such as a disease, a beneficial response to anagent acting on the leukotriene pathway or side-effects linked to atreatment with this agent, are then verified by screening a largerpopulation of trait positive and trait negative individuals usingpolymorphism analysis techniques such as the techniques described above.Polymorphisms which exhibit a statistically significant correlation withthe detectable phenotype are deemed responsible for the detectablephenotype.

Most of the biallelic polymorphisms of the FLAP gene observed in thecontext of the present invention do not appear to drastically modify theamino acid sequence of the FLAP protein. Also, they do not seem to belocated in splicing sequences. However, they may be associated withchanges in basic FLAP expression in one or more tissues. Suchpolymorphisms may eventually modify the transcription rate of FLAP DNA,FLAP in RNA stability, or the translation rate of FLAP mRNA.

The biallelic polymorphisms may also be associated with changes in themodulation of FLAP expression through expression modifiers. The term“expression modifier” is intended to encompass chemical agents thatmodulate the action of FLAP through modulation of FLAP gene expression.

The basic FLAP expression levels in different tissues can be determinedby analyses of tissue samples from individuals typed for the presence orabsence of a specific polymorphism. Any convenient method can be usedsuch as ELISA, RIA for protein quantitation, and such as Northern blotor other hybridization analyses, and quantitative RT-PCR for mRNAquantitation. The tissue specific expression can then be correlated withthe genotype. More details on some of these methods are provided belowunder the heading “Screening of agents”.

Furthermore, the strong association observed for the first time betweenthe FLAP gene and asthma confirms the need to locate and study anymutation of the FLAP gene as such mutation is susceptible of having anincidence on leukotriene metabolism and hence on the therapeutic choicesmade when considering various treatment alternatives for an individualwith a particular condition involving the leukotriene pathway.

There are numerous possibilities for causal mutations within the FLAPgene. One of the causal mutations can be an amino acid change in theFLAP protein which can lead to alterations in FLAP substrate specificityand/or activity. Methods for analyzing protein-protein or protein-ligandinteractions are detailed below under the heading “Screening of agents”.

Another possible causal mutation of the FLAP gene is a modification inits regulatory region, and particularly in the sequence of its nativepromoter. This type of mutation can be studied through the determinationof basic expression levels by expression assays for the particularpromoter sequence. The assays may be performed with the FLAP codingsequence or with a detectable marker sequence. To determine tissuespecificity, the assay is performed in cells from different sources.Some methods are discussed in more detail below under the heading“Screening of agents”.

When used herein, the term “basic expression levels” intends todesignate FLAP expression levels normally observed in individuals notbearing the associated allele of biallelic markers of the presentinvention.

In another embodiment, the mutant FLAP allele which causes a detectablephenotype can be isolated by obtaining a nucleic acid sample such as agenomic library or a cDNA library from an individual expressing thedetectable phenotype. The nucleic acid sample can be contacted with oneor more probes lying in the region of the FLAP gene where the associatedbiallelic marker or group of biallelic markers or with PCR-typeableprimers specific to the amplification of this biallelic marker or groupof biallelic markers. The mutation can be identified by conductingsequencing reactions on the nucleic acids which hybridize with theprobes defined herein or which show amplification by PCR. The region ofthe FLAP gene containing the mutation responsible for the detectablephenotype may be used in diagnostic techniques such as those describedbelow. For example, microsequencing oligonucleotides, oroligonucleotides containing the mutation responsible for the detectablephenotype for amplification, or hybridization based diagnostics, such asthose described herein, may be used for detecting individuals sufferingfrom the detectable phenotype or individuals at risk of developing thedetectable phenotype at a subsequent time. In addition, the FLAP alleleresponsible for the detectable phenotype may be used in gene therapy.The FLAP allele responsible for the detectable phenotype may also becloned into an expression vector to express the mutant FLAP protein asdescribed herein.

XIII. Biallelic Markers of The Invention in Methods of GeneticDiagnostics

The biallelic markers of the present invention can also be used todevelop diagnostics tests capable of identifying individuals who expressa detectable trait as the result of a specific genotype or individualswhose genotype places them at risk of developing a detectable trait at asubsequent time. The trait analyzed using the present diagnostics may beany detectable trait, including a disease involving the leukotrienepathway, a beneficial response to treatment with agents acting on theleukotriene pathway or side-effects related to treatment with agentsacting on the leukotriene pathway.

The diagnostic techniques of the present invention may employ a varietyof methodologies to determine whether a test subject has a biallelicmarker pattern associated with an increased risk of developing adetectable trait or whether the individual suffers from a detectabletrait as a result of a particular mutation, including methods whichenable the analysis of individual chromosomes for haplotyping, such asfamily studies, single sperm DNA analysis or somatic hybrids.

The present invention provides diagnostic methods to determine whetheran individual is at risk of developing a disease or suffers from adisease resulting from a mutation or a polymorphism in the FLAP gene.The present invention also provides methods to determine whether anindividual is likely to respond positively to an agent acting on theleukotriene pathway or whether an individual is at risk of developing anadverse side-effect to an agent acting on the leukotriene pathway.

These methods involve obtaining a nucleic acid sample from theindividual and, determining, whether the nucleic acid sample contains atleast one allele or at least one biallelic marker haplotype, indicativeof a risk of developing the trait or indicative that the individualexpresses the trait as a result of possessing a particular FLAPpolymorphism or mutation (trait-causing allele).

Preferably, in such diagnostic methods, a nucleic acid sample isobtained from the individual and this sample is genotyped using methodsdescribed above in VIII. The diagnostics may be based on a singlebiallelic marker or on a group of biallelic markers.

In each of these methods, a nucleic acid sample is obtained from thetest subject and the biallelic marker pattern of one or more of thebiallelic markers A1 to A28, the complements thereof or a biallelicmarker in linkage disequilibrium therewith is determined.

In one embodiment, a PCR amplification is conducted on the nucleic acidsample to amplify regions in which polymorphisms associated with adetectable phenotype have been identified. The amplification productsare sequenced to determine whether the individual possesses one or moreFLAP polymorphisms associated with a detectable phenotype. The primersused to generate amplification products may comprise the primers B1 toB17 and C1 to C17. Alternatively, the nucleic acid sample is subjectedto microsequencing reactions as described above to determine whether theindividual possesses one or more FLAP polymorphisms associated with adetectable phenotype resulting from a mutation or a polymorphism in theFLAP gene. The primers used in the microsequencing reactions may includethe primers D1 to D28 and E1 to E28. In another embodiment, the nucleicacid sample is contacted with one or more allele specificoligonucleotide probes which, specifically hybridize to one or more FLAPalleles associated with a detectable phenotype. The probes used in thehybridization assay may include the probes P1 to P28, a complementarysequence thereto or a fragment thereof comprising the polymorphic base.In another embodiment, the nucleic acid sample is contacted with asecond FLAP oligonucleotide capable of producing an amplificationproduct when used with the allele specific oligonucleotide in anamplification reaction. The presence of an amplification product in theamplification reaction indicates that the individual possesses one ormore FLAP alleles associated with a detectable phenotype.

In a preferred embodiment, the identity of the nucleotide present at, atleast one biallelic marker selected from the group consisting of A2,A14, A16, A18, A19, A22, and A23, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith, isdetermined and the detectable trait is asthma. In another preferredembodiment the identity of the nucleotide present at, at least one ofthe polymorphic sites selected from the group consisting of A14 and A19,and the complements thereof, or optionally the biallelic markers inlinkage disequilibrium therewith, is determined. In more preferredembodiment, the identity of the nucleotide present at the polymorphicsite A19, and the complements thereof, or optionally the biallelicmarkers in linkage disequilibrium therewith, is determined. Diagnostickits comprising polynucleotides of the present invention are furtherdescribed in the present invention.

These diagnostic methods are extremely valuable as they can, in certaincircumstances, be used to initiate preventive treatments or to allow anindividual carrying a significant haplotype to foresee warning signssuch as minor symptoms. In diseases in which attacks may be extremelyviolent and sometimes fatal if not treated on time, such as asthma, theknowledge of a potential predisposition, even if this predisposition isnot absolute, might contribute in a very significant manner to treatmentefficacy.

The present invention also encompasses diagnostic kits comprising one ormore polynucleotides of the invention with a portion or all of thenecessary reagents and instructions for genotyping a test subject bydetermining the identity of a nucleotide at a FLAP-related biallelicmarker. The polynucleotides of a kit may optionally be attached to asolid support, or be part of an array or addressable array ofpolynucleotides. The kit may provide for the determination of theidentity of the nucleotide at a marker position by any method known inthe art including, but not limited to, a sequencing assay method, amicrosequencing assay method, a hybridization assay method, or amismatch detection assay based on polymerases and/or ligases. Thediagnostic kits can be manufactured to perform any of the genotypingmethods described in the current application using manufacturing andformulation methods commonly in the art. Preferably such a kit mayprovide for the determination of the allele of a biallelic markerselected from FLAP-related biallelic markers. Optionally such a kit mayinclude instructions for scoring the results of the determination withrespect to the test subjects' risk of contracting a disease involvingthe leukotriene pathway, a beneficial response to treatment with agentsacting on the leukotriene pathway or side-effects related to treatmentwith agents acting on the leukotriene pathway.

XIV. Treatment of Diseases Involving the Leukotriene Pathway

The invention also relates to a method of determining whether a subjectis likely to respond positively to treatment with a medicament,preferably a medicament acting directly or indirectly on the leukotrienepathway.

The method comprises identifying a first population of individuals whoresponse positively to said medicament and a second population ofindividuals who respond negatively to said medicament. One or morebiallelic markers is identified in the first population which isassociated with a positive response to said medicament or one or morebiallelic markers is identified in the second population which isassociated with a negative response to said medicament. The biallelicmarkers may be identified using the techniques described herein.

The DNA sample is then obtained form the subject tested. The DNA sampleis analyzed to determine whether it comprises one or more alleles ofbiallelic markers associated with a positive response to a medicament orone or more alleles of biallelic markers associated with a negativeresponse to treatment with the medicament. In some embodiments, the DNAsample is analyzed to identify subjects whose DNA comprises one or morealleles of biallelic markers associated with a positive response to themedicament and whose DNA lacks one or more alleles of biallelic markersassociated with a negative response to treatment with the medicament.

In other embodiments, the medicament is administered to the subject in aclinical trial if the DNA sample contains one or more alleles ofbiallelic markers associated with positive response to the medicamentand/or if the DNA sample lacks one or more alleles of biallelic markersassociated with a negative response to treatment with the medicament. Inpreferred embodiments, the medicament is an anti-asthma drug such asZileuton. In other embodiments, the negative response comprises one ormore side-effects, such as increased liver transaminase levels. Usingthe methods of the present invention, the evaluation of drug efficacymay be conducted in a population of individuals likely to respondfavorably to the medicament.

The invention also concerns a method for the clinical testing of amedicament, preferably a medicament acting directly or indirectly on theleukotriene pathway. The method comprises the following steps: a)administering a medicament, preferably a medicament capable of actingdirectly or indirectly on the leukotriene pathway to a heterogeneouspopulation of individuals; b) identifying a first population ofindividuals who response positively to said medicament and a secondpopulation of individuals who respond negatively to said medicament; c)identifying biallelic markers in said first population which areassociated with a positive response to said medicament and/or biallelicmarkers in said second population which are associated with a negativeresponse to said medicament; d) selecting individuals whose DNAcomprises one or more alleles of biallelic markers associated with apositive response to said medicament and/or whose DNA lacks one or morealleles of biallelic markers associated with a negative response to saidmedicament; and, d) administering said medicament to said individuals.

Such methods are deemed to be extremely useful to increase thebenefit/risk ratio resulting from the administration of medicamentswhich may cause undesirable side-effects and/or be inefficacious to aportion of the patient population to which it is normally administered.

Once an individual has been diagnosed as suffering from a diseaseinvolving the leukotriene pathway such as asthma, selection tests arecarried out to determine whether the DNA of this individual comprisesalleles of a biallelic marker or of a group of biallelic markersassociated a positive response to treatment or with a negative responseto treatment which may include either side-effects or unresponsiveness.

The selection of the patient to be treated using the method of thepresent invention can be carried out through the detection methodsdescribed above. The individuals which are to be selected are preferablythose whose DNA does not comprise alleles of a biallelic marker or of agroup of biallelic markers associated with negative response totreatment.

Once the patient's genetic predispositions have been determined, theclinician can select appropriate treatment for which the particularside-effect observed for the patient has not been reported or has beenreported only marginally and preferably from an allelic associationwhich does not involve the same biallelic marker or markers as thosefound in the DNA of the patient. Several drugs useful in the treatmentof diseases involving the leukotriene pathway may be chosen. Compoundsacting on the leukotriene pathway are described for example in U.S. Pat.Nos. 4,873,259; 4,970,215; 5,310,744; 5,225,421; and 5,081,138, or in EP0 419 049, the disclosures of which are incorporated by reference.

XV. FLAP Proteins and Polypeptide Fragments

The term “FLAP polypeptides” is used herein to embrace all of theproteins and polypeptides of the present invention. Also forming part ofthe invention are polypeptides encoded by the polynucleotides of theinvention, as well as fusion polypeptides comprising such polypeptides.The invention embodies FLAP proteins from humans, including isolated orpurified FLAP proteins consisting, consisting essentially, or comprisingthe sequence of SEQ ID NO: 3 and comprising an isoleucine at position127 in SEQ ID NO: 3. It should be noted the FLAP proteins of theinvention are based on the naturally-occurring variant of the amino acidsequence of human FLAP, wherein the valine residue of amino acidposition 127 in SEQ ID NO: 3 has been replaced with an isoleucineresidue. This variant protein and the fragments thereof which containamino acid position 127 of SEQ ID NO: 3 are collectively referred toherein as “127-Ile variants” or 127-Ile FLAP polypeptides”.

The present invention embodies isolated, purified, and recombinantpolypeptides comprising a contiguous span of at least 6 amino acids,preferably at least 8 to 10 amino acids, more preferably at least 12,15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID NO: 3, wherein saidcontiguous span includes an isoleucine residue at amino acid position127 in SEQ ID NO: 3. In other preferred embodiments the contiguousstretch of amino acids comprises the site of a mutation or functionalmutation, including a deletion, addition, swap or truncation of theamino acids in the FLAP protein sequence.

FLAP proteins are preferably isolated from human or mammalian tissuesamples or expressed from human or mammalian genes. The FLAPpolypeptides of the invention can be made using routine expressionmethods known in the art. The polynucleotide encoding the desiredpolypeptide, is ligated into an expression vector suitable for anyconvenient host. Both eukaryotic and prokaryotic host systems are usedin forming recombinant polypeptides, and a summary of some of the morecommon systems. The polypeptide is then isolated from lysed cells orfrom the culture medium and purified to the extent needed for itsintended use. Purification is by any technique known in the art, forexample, differential extraction, salt fractionation, chromatography,centrifugation, and the like. See, for example, Methods in Enzymologyfor a variety of methods for purifying proteins.

In addition, shorter protein fragments are produced by chemicalsynthesis. Alternatively the proteins of the invention are extractedfrom cells or tissues of humans or non-human animals. Methods forpurifying proteins are known in the art, and include the use ofdetergents or chaotropic agents to disrupt particles followed bydifferential extraction and separation of the polypeptides by ionexchange chromatography, affinity chromatography, sedimentationaccording to density, and gel electrophoresis.

Any FLAP cDNA, including SEQ ID NO: 2, is used to express FLAP proteinsand polypeptides. The preferred FLAP cDNA comprises the allele A of thebiallelic marker A21. The nucleic acid encoding the FLAP protein orpolypeptide to be expressed is operably linked to a promoter in anexpression vector using conventional cloning technology. The FLAP insertin the expression vector may comprise the full coding sequence for theFLAP protein or a portion thereof. For example, the FLAP derived insertmay encode a polypeptide comprising at least 10 consecutive amino acidsof the FLAP protein of SEQ ID NO: 3, where in said consecutive aminoacids comprising an isoleucine residue in amino acid position 127.

The expression vector is any of the mammalian, yeast, insect orbacterial expression systems known in the art. Commercially availablevectors and expression systems are available from a variety of suppliersincluding Genetics Institute (Cambridge, Mass.), Stratagene (La Jolla,Calif.), Promega (Madison, Wis.), and Invitrogen (San Diego, Calif.). Ifdesired, to enhance expression and facilitate proper protein folding,the codon context and codon pairing of the sequence is optimized for theparticular expression organism in which the expression vector isintroduced, as explained by Hatfield, et al., U.S. Pat. No. 5,082,767.

In one embodiment, the entire coding sequence of the FLAP cDNA throughthe poly A signal of the cDNA are operably linked to a promoter in theexpression vector. Alternatively, if the nucleic acid encoding a portionof the FLAP protein lacks a methionine to serve as the initiation site,an initiating methionine can be introduced next to the first codon ofthe nucleic acid using conventional techniques. Similarly, if the insertfrom the FLAP cDNA lacks a poly A signal, this sequence can be added tothe construct by, for example, splicing out the Poly A signal from pSG5(Stratagene) using BglI and SalI restriction endonuclease enzymes andincorporating it into the mammalian expression vector pXT1 (Stratagene).pXT1 contains the LTRs and a portion of the gag gene from Moloney MurineLeukemia Virus. The position of the LTRs in the construct allowefficient stable transfection. The vector includes the Herpes SimplexThymidine Kinase promoter and the selectable neomycin gene. The nucleicacid encoding the FLAP protein or a portion thereof is obtained by PCRfrom a bacterial vector containing the FLAP cDNA of SEQ ID NO: 3 usingoligonucleotide primers complementary to the FLAP cDNA or portionthereof and containing restriction endonuclease sequences for Pst Iincorporated into the 5′primer and BglII at the 5′ end of thecorresponding cDNA 3′ primer, taking care to ensure that the sequenceencoding the FLAP protein or a portion thereof is positioned properlywith respect to the poly A signal. The purified fragment obtained fromthe resulting PCR reaction is digested with PstI, blunt ended with anexonuclease, digested with Bgl II, purified and ligated to pXT1, nowcontaining a poly A signal and digested with BglII.

The ligated product is transfected into mouse NIH 3T3 cells usingLipofectin (Life Technologies, Inc., Grand Island, N.Y.) underconditions outlined in the product specification. Positive transfectantsare selected after growing the transfected cells in 600 ug/ml G418(Sigma, St. Louis, Mo.).

Alternatively, the nucleic acids encoding the FLAP protein or a portionthereof is cloned into pED6dpc2 (Genetics Institute, Cambridge, Mass.).The resulting pED6dpc2 constructs is transfected into a suitable hostcell, such as COS 1 cells. Methotrexate resistant cells are selected andexpanded.

The above procedures may also be used to express a mutant FLAP proteinresponsible for a detectable phenotype or a portion thereof.

The expressed proteins are purified using conventional purificationtechniques such as ammonium sulfate precipitation or chromatographicseparation based on size or charge. The protein encoded by the nucleicacid insert may also be purified using standard immunochromatographytechniques. In such procedures, a solution containing the expressed FLAPprotein or portion thereof, such as a cell extract, is applied to acolumn having antibodies against the FLAP protein or portion thereof isattached to the chromatography matrix. The expressed protein is allowedto bind the immunochromatography column. Thereafter, the column iswashed to remove non-specifically bound proteins. The specifically boundexpressed protein is then released from the column and recovered usingstandard techniques.

To confirm expression of the FLAP protein or a portion thereof, theproteins expressed from host cells containing an expression vectorcontaining an insert encoding the FLAP protein or a portion thereof canbe compared to the proteins expressed in host cells containing theexpression vector without an insert. The presence of a band in samplesfrom cells containing the expression vector with an insert which isabsent in samples from cells containing the expression vector without aninsert indicates that the FLAP protein or a portion thereof is beingexpressed. Generally, the band will have the mobility expected for theFLAP protein or portion thereof. However, the band may have a mobilitydifferent than that expected as a result of modifications such asglycosylation, ubiquitination, or enzymatic cleavage.

Antibodies capable of specifically recognizing the expressed FLAPprotein or a portion thereof are described below.

If antibody production is not possible, the nucleic acids encoding theFLAP protein or a portion thereof is incorporated into expressionvectors designed for use in purification schemes employing chimericpolypeptides. In such strategies the nucleic acid encoding the FLAPprotein or a portion thereof is inserted in frame with the gene encodingthe other half of the chimera. The other half of the chimera is β-globinor a nickel binding polypeptide encoding sequence. A chromatographymatrix having antibody to β-globin or nickel attached thereto is thenused to purify the chimeric protein. Protease cleavage sites isengineered between the β-globin gene or the nickel binding polypeptideand the FLAP protein or portion thereof. Thus, the two polypeptides ofthe chimera is separated from one another by protease digestion.

One useful expression vector for generating β-globin chimerics is pSG5(Stratagene), which encodes rabbit β-globin. Intron II of the rabbitβ-globin gene facilitates splicing of the expressed transcript, and thepolyadenylation signal incorporated into the construct increases thelevel of expression. These techniques are well known to those skilled inthe art of molecular biology. Standard methods are published in methodstexts such as Davis et al., (1986) and many of the methods are availablefrom Stratagene, Life Technologies, Inc., or Promega. Polypeptide mayadditionally be produced from the construct using in vitro translationsystems such as the In vitro Express™ Translation Kit (Stratagene).

Antibodies that Bind FLAP Polypeptides of the Invention

Any FLAP polypeptide or whole protein may be used to generate antibodiescapable of specifically binding to expressed FLAP protein or fragmentsthereof as described. The antibody compositions of the invention arecapable of specifically binding or specifically bind to the 127-Ilevariant of the FLAP protein. For an antibody composition to specificallybind to the 127-Ile variant of FLAP it must demonstrate at least a 5%,10%, 15%, 20%, 25%, 50%, or 100% greater binding affinity for fulllength 127-Ile variant of FLAP than for full length 127-Val variant ofFLAP in an ELISA, RIA, or other antibody-based binding assay.

In a preferred embodiment of the invention antibody compositions arecapable of selectively binding, or selectively bind to anepitope-containing fragment of a polypeptide comprising a contiguousspan of at least 6 amino acids, preferably at least 8 to 10 amino acids,more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acidsof SEQ ID NO: 3, wherein said epitope comprises an isoleucine residue atamino acid position 127 in SEQ ID NO: 3, wherein said antibodycomposition is optionally either polyclonal or monoclonal.

The present invention also contemplates the use of polypeptidescomprising a contiguous span of at least 6 amino acids, preferably atleast 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 50,or 100 amino acids of a FLAP polypeptide in the manufacture ofantibodies, wherein said contiguous span comprises an isoleucine residueat amino acid position 127 of SEQ ID NO: 3. In a preferred embodimentsuch polypeptides are useful in the manufacture of antibodies to detectthe presence and absence of the 127-Ile variant.

Non-human animals or mammals, whether wild-type or transgenic, whichexpress a different species of FLAP than the one to which antibodybinding is desired, and animals which do not express FLAP (i.e. a FLAPknock out animal as described in herein) are particularly useful forpreparing antibodies. FLAP knock out animals will recognize all or mostof the exposed regions of FLAP as foreign antigens, and thereforeproduce antibodies with a wider array of FLAP epitopes. Moreover,smaller polypeptides with only 10 to 30 amino acids may be useful inobtaining specific binding to the 127-Ile variant. In addition, thehumoral immune system of animals which produce a species of FLAP thatresembles the antigenic sequence will preferentially recognize thedifferences between the animal's native FLAP species and the antigensequence, and produce antibodies to these unique sites in the antigensequence. Such a technique will be particularly useful in obtainingantibodies that specifically bind to the 127-Ile variant.

XVI. Recombinant Vectors, Cell Hosts, and Transgenic Animals

Recombinant Vectors

The term “vector” is used herein to designate either a circular or alinear DNA or RNA molecule, which is either double-stranded orsingle-stranded, and which comprise at least one polynucleotide ofinterest that is sought to be transferred in a cell host or in aunicellular or multicellular host organism.

The present invention encompasses a family of recombinant vectors thatcomprise a regulatory polynucleotide derived from the FLAP genomicsequence, or a coding polynucleotide from the FLAP genomic sequence.Consequently, the present invention further deals with a recombinantvector comprising either a regulatory polynucleotide comprised in thenucleic acid of SEQ ID NO: 1 or a polynucleotide comprising the FLAPcoding sequence or both.

Generally, a recombinant vector of the invention may comprise any of thepolynucleotides described herein, including regulatory sequences andcoding sequences, as well as any FLAP primer or probe as defined above.

In a first preferred embodiment, a recombinant vector of the inventionis used to amplify the inserted polynucleotide derived from a FLAPgenomic sequence of SEQ ID NO: 1 or a FLAP cDNA, for example the cDNA ofSEQ ID NO: 2 in a suitable cell host, this polynucleotide beingamplified at every time that the recombinant vector replicates.

A second preferred embodiment of the recombinant vectors according tothe invention consists of expression vectors comprising either aregulatory polynucleotide or a coding nucleic acid of the invention, orboth. Within certain embodiments, expression vectors are employed toexpress the FLAP polypeptide which can be then purified and, for examplebe used in ligand screening assays or as an immunogen in order to raisespecific antibodies directed against the FLAP protein. In otherembodiments, the expression vectors are used for constructing transgenicanimals and also for gene therapy. Expression requires that appropriatesignals are provided in the vectors, said signals including variousregulatory elements, such as enhancers/promoters from both viral andmammalian sources that drive expression of the genes of interest in hostcells. Dominant drug selection markers for establishing permanent,stable cell clones expressing the products are generally included in theexpression vectors of the invention, as they are elements that linkexpression of the drug selection markers to expression of thepolypeptide.

More particularly, the present invention relates to expression vectorswhich include nucleic acids encoding a FLAP protein, preferably the FLAPprotein of the amino acid sequence of SEQ ID NO: 3, more preferably theFLAP protein of the amino acid sequence of SEQ ID NO: 3 bearing anisoleucine residues in position 127 or variants or fragments thereof,under the control of a regulatory sequence selected among the FLAPregulatory polynucleotides, or alternatively under the control of anexogenous regulatory sequence.

Consequently, preferred expression vectors of the invention are selectedfrom the group consisting of: (a) the FLAP regulatory sequence comprisedtherein drives the expression of a coding polynucleotide operably linkedthereto; (b) the FLAP coding sequence is operably linked to regulationsequences allowing its expression in a suitable cell host and/or hostorganism.

Recombinant vectors comprising a nucleic acid containing a FLAP-relatedbiallelic marker is also part of the invention. In a preferredembodiment, said biallelic marker is selected from the group consistingof A1 to A28, and the complements thereof, or optionally the biallelicmarkers in linkage disequilibrium therewith. Optionally, said biallelicmarker is selected from the group consisting of A1 to A13, A15, A17 toA28, and the complements thereof, or optionally the biallelic markers inlinkage disequilibrium therewith.

Some of the elements which can be found in the vectors of the presentinvention are described in further detail in the following sections.

1. General Features of the Expression Vectors of the Invention

A recombinant vector according to the invention comprises, but is notlimited to, a YAC (Yeast Artificial Chromosome), a BAC (BacterialArtificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or evena linear DNA molecule which may consist of a chromosomal,non-chromosomal, semi-synthetic and synthetic DNA. Such a recombinantvector can comprise a transcriptional unit comprising an assembly of:

(1) a genetic element or elements having a regulatory role in geneexpression, for example promoters or enhancers. Enhancers are cis-actingelements of DNA, usually from about 10 to 300 bp in length that act onthe promoter to increase the transcription.

(2) a structural or coding sequence which is transcribed into mRNA andeventually translated into a polypeptide, said structural or codingsequence being operably linked to the regulatory elements described in(1); and

(3) appropriate transcription initiation and termination sequences.Structural units intended for use in yeast or eukaryotic expressionsystems preferably include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, when arecombinant protein is expressed without a leader or transport sequence,it may include a N-terminal residue. This residue may or may not besubsequently cleaved from the expressed recombinant protein to provide afinal product.

Generally, recombinant expression vectors will include origins ofreplication, selectable markers permitting transformation of the hostcell, and a promoter derived from a highly expressed gene to directtranscription of a downstream structural sequence. The heterologousstructural sequence is assembled in appropriate phase with translationinitiation and termination sequences, and preferably a leader sequencecapable of directing secretion of the translated protein into theperiplasmic space or the extracellular medium. In a specific embodimentwherein the vector is adapted for transfecting and expressing desiredsequences in mammalian host cells, preferred vectors will comprise anorigin of replication in the desired host, a suitable promoter andenhancer, and also any necessary ribosome binding sites, polyadenylationsite, splice donor and acceptor sites, transcriptional terminationsequences, and 5′-flanking non-transcribed sequences. DNA sequencesderived from the SV40 viral genome, for example SV40 origin, earlypromoter, enhancer, splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

The in vivo expression of a FLAP polypeptide of SEQ ID NO: 3 orfragments or variants thereof may be useful in order to correct agenetic defect related to the expression of the native gene in a hostorganism or to the production of a biologically inactive FLAP protein.

Consequently, the present invention also deals with recombinantexpression vectors mainly designed for the in vivo production of theFLAP polypeptide of SEQ ID NO: 3 or fragments or variants thereof by theintroduction of the appropriate genetic material in the organism of thepatient to be treated. This genetic material may be introduced in vitroin a cell that has been previously extracted from the organism, themodified cell being subsequently reintroduced in the said organism,directly in vivo into the appropriate tissue.

2. Regulatory Elements

Promoters

The suitable promoter regions used in the expression vectors accordingto the present invention are chosen taking into account the cell host inwhich the heterologous gene has to be expressed. The particular promoteremployed to control the expression of a nucleic acid sequence ofinterest is not believed to be important, so long as it is capable ofdirecting the expression of the nucleic acid in the targeted cell. Thus,where a human cell is targeted, it is preferable to position the nucleicacid coding region adjacent to and under the control of a promoter thatis capable of being expressed in a human cell, such as, for example, ahuman or a viral promoter.

A suitable promoter may be heterologous with respect to the nucleic acidfor which it controls the expression or alternatively can be endogenousto the native polynucleotide containing the coding sequence to beexpressed. Additionally, the promoter is generally heterologous withrespect to the recombinant vector sequences within which the constructpromoter/coding sequence has been inserted.

Promoter regions can be selected from any desired gene using, forexample, CAT (chloramphenicol transferase) vectors and more preferablypKK232-8 and pCM7 vectors.

Preferred bacterial promoters are the LacI, LacZ, the T3 or T7bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and trppromoters (EP 0036776), the polyhedrin promoter, or the p10 proteinpromoter from baculovirus (Kit Novagen) (Smith et al., 1983; O'Reilly etal., 1992), the lambda PR promoter or also the trc promoter.

Eukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein-L.Selection of a convenient vector and promoter is well within the levelof ordinary skill in the art.

The choice of a promoter is well within the ability of a person skilledin the field of genetic engineering. For example, one may refer to thebook of Sambrook et al. (1989) or also to the procedures described byFuller et al. (1996).

Other Regulatory Elements

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

The vector containing the appropriate DNA sequence as described above,more preferably FLAP gene regulatory polynucleotide, a polynucleotideencoding the FLAP polypeptide selected from the group consisting of SEQID NO: 1 or a fragment or a variant thereof and SEQ ID NO: 2, or both ofthem, can be utilized to transform an appropriate host to allow theexpression of the desired polypeptide or polynucleotide.

3. Selectable Markers

Such markers would confer an identifiable change to the cell permittingeasy identification of cells containing the expression construct. Theselectable marker genes for selection of transformed host cells arepreferably dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, TRP1 for S. cerevisiae or tetracycline, rifampicin orampicillin resistance in E. coli, or levan saccharase for mycobacteria,this latter marker being a negative selection marker.

4. Preferred Vectors.

Bacterial Vectors

As a representative but non-limiting example, useful expression vectorsfor bacterial use can comprise a selectable marker and a bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of pBR322 (ATCC 37017). Such commercialvectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), andGEM1 (Promega Biotec, Madison, Wis., USA).

Large numbers of other suitable vectors are known to those of skill inthe art, and commercially available, such as the following bacterialvectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174,pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene);ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); pWLNEO, pSV2CAT,pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia);pQE-30 (QIAexpress).

Bacteriophage Vectors

The PI bacteriophage vector may contain large inserts ranging from about80 to about 100 kb.

The construction of PI bacteriophage vectors such as p158 or p158/neo8are notably described by Sternberg (1992, 1994). Recombinant PI clonescomprising FLAP nucleotide sequences may be designed for inserting largepolynucleotides of more than 40 kb (Linton et al., 1993). To generate P1DNA for transgenic experiments, a preferred protocol is the protocoldescribed by McCormick et al. (1994).

Baculovirus Vectors

A suitable vector for the expression of the FLAP polypeptide of SEQ IDNO: 6 or fragments or variants thereof is a baculovirus vector that canbe propagated in insect cells and in insect cell lines. A specificsuitable host vector system is the pVL1392/1393 baculovirus transfervector (Pharmingen) that is used to transfect the SF9 cell line (ATCCNo. CRL 1711) which is derived from Spodoptera frugiperda.

Other suitable vectors for the expression of the FLAP polypeptide of SEQID NO: 6 or fragments or variants thereof in a baculovirus expressionsystem include those described by Chai et al. (1993), Vlasak et al.(1983) and Lenhard et al. (1996).

Viral Vectors

In one specific embodiment, the vector is derived from an adenovirus.Preferred adenovirus vectors according to the invention are thosedescribed by Feldman and Steg (1996) or Ohno et al. (1994). Anotherpreferred recombinant adenovirus according to this specific embodimentof the present invention is the human adenovirus type 2 or 5 (Ad 2 or Ad5) or an adenovirus of animal origin (French patent application No.FR-93. 05954).

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery systems of choice for thetransfer of exogenous polynucleotides in vivo, particularly to mammals,including humans. These vectors provide efficient delivery of genes intocells, and the transferred nucleic acids are stably integrated into thechromosomal DNA of the host.

Particularly preferred retroviruses for the preparation or constructionof retroviral in vitro or in vitro gene delivery vehicles of the presentinvention include retroviruses selected from the group consisting ofMink-Cell Focus Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis virus and Rous Sarcoma virus. Particularlypreferred Murine Leukemia Viruses include the 4070A and the 1504Aviruses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCCNo VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Virus(ATCC No VR-190; PCT Application No WO 94/24298). Particularly preferredRous Sarcoma Viruses include Bryan high titer (ATCC Nos. VR-334, VR-657,VR-726, VR-659 and VR-728). Other preferred retroviral vectors are thosedescribed in Roth et al. (1996), PCT Application No WO 93/25234, PCTApplication No WO 94/06920, Roux et al., 1989, Julan et al., 1992 andNeda et al., 1991.

Yet another viral vector system that is contemplated by the inventionconsists in the adeno-associated virus (AAV). The adeno-associated virusis a naturally occurring defective virus that requires another virus,such as an adenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle (Muzyczka et al., 1992). It isalso one of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (Flotte etal., 1992; Samulski et al., 1989; McLaughlin et al., 1989). Oneadvantageous feature of AAV derives from its reduced efficacy fortransducing primary cells relative to transformed cells.

BAC Vectors

The bacterial artificial chromosome (BAC) cloning system (Shizuya etal., 1992) has been developed to stably maintain large fragments ofgenomic DNA (100-300 kb) in E. coli. A preferred BAC vector consists ofpBeloBAC11 vector that has been described by Kim et al. (1996). BAClibraries are prepared with this vector using size-selected genomic DNAthat has been partially digested using enzymes that permit ligation intoeither the Bam HI or HindIII sites in the vector. Flanking these cloningsites are T7 and SP6 RNA polymerase transcription initiation sites thatcan be used to generate end probes by either RNA transcription or PCRmethods. After the construction of a BAC library in E. Coli, BAC DNA ispurified from the host cell as a supercoiled circle. Converting thesecircular molecules into a linear form precedes both size determinationand introduction of the BACs into recipient cells. The cloning site isflanked by two NotI sites, permitting cloned segments to be excised fromthe vector by Not I digestion. Alternatively, the DNA insert containedin the pBeloBAC11 vector may be linearized by treatment of the BACvector with the commercially available enzyme lambda terminase thatleads to the cleavage at the unique cosN site, but this cleavage methodresults in a full length BAC clone containing both the insert DNA andthe BAC sequences.

5. Delivery of the Recombinant Vectors

In order to effect expression of the polynucleotides and polynucleotideconstructs of the invention, these constructs must be delivered into acell. This delivery may be accomplished in vitro, as in laboratoryprocedures for transforming cell lines, or in vivo or ex vivo, as in thetreatment of certain diseases states.

One mechanism is viral infection where the expression construct isencapsulated in an infectious viral particle.

Several non-viral methods for the transfer of polynucleotides intocultured mammalian cells are also contemplated by the present invention,and include, without being limited to, calcium phosphate precipitation(Graham et al., 1973; Chen et al., 1987;), DEAE-dextran (Gopal, 1985),electroporation (Tur-Kaspa et al., 1986; Potteret al., 1984), directmicroinjection (Harland et al., 1985), DNA-loaded liposomes (Nicolau etal., 1982; Fraley et al., 1979), and receptor-mediate transfection (Wuand Wu, 1987; 1988). Some of these techniques may be successfullyadapted for in vivo or ex vivo use.

Once the expression polynucleotide has been delivered into the cell, itmay be stably integrated into the genome of the recipient cell. Thisintegration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle.

One specific embodiment for a method for delivering a protein or peptideto the interior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a physiologically acceptablecarrier and a naked polynucleotide operatively coding for thepolypeptide of interest into the interstitial space of a tissuecomprising the cell, whereby the naked polynucleotide is taken up intothe interior of the cell and has a physiological effect. This isparticularly applicable for transfer in vitro but it may be applied toin vivo as well.

Compositions for use in vitro and in vivo comprising a “naked”polynucleotide are described in PCT application No. WO 90/11092 (VicalInc.) and also in PCT application No WO 95/11307 (Institut Pasteur,INSERM, Université d'Ottawa) as well as in the articles of Tacson et al.(1996) and of Huygen et al. (1996).

In still another embodiment of the invention, the transfer of a nakedpolynucleotide of the invention, including a polynucleotide construct ofthe invention, into cells may be proceeded with a particle bombardment(biolistic), said particles being DNA-coated microprojectilesaccelerated to a high velocity allowing them to pierce cell membranesand enter cells without killing them, such as described by Klein et al.(1987).

In a further embodiment, the polynucleotide of the invention may beentrapped in a liposome (Ghosh and Bacchawat, 1991; Wong et al., 1980;Nicolau et al., 1987)

In a specific embodiment, the invention provides a composition for thein vivo production of the FLAP protein or polypeptide described herein.It comprises a naked polynucleotide operatively coding for thispolypeptide, in solution in a physiologically acceptable carrier, andsuitable for introduction into a tissue to cause cells of the tissue toexpress the said protein or polypeptide.

The amount of vector to be injected to the desired host organism variesaccording to the site of injection. As an indicative dose, it will beinjected between 0.1 and 100 μg of the vector in an animal body,preferably a mammal body, for example a mouse body.

In another embodiment of the vector according to the invention, it maybe introduced in vitro in a host cell, preferably in a host cellpreviously harvested from the animal to be treated and more preferably asomatic cell such as a muscle cell. In a subsequent step, the cell thathas been transformed with the vector coding for the desired FLAPpolypeptide or the desired fragment thereof is reintroduced into theanimal body in order to deliver the recombinant protein within the bodyeither locally or systemically.

Cell Hosts

Another object of the invention consists of a host cell that has beentransformed or transfected with one of the polynucleotides describedtherein. Are included host cells that are transformed (prokaryoticcells) or that are transfected (eukaryotic cells) with a recombinantvector such as one of those described above.

Generally, a recombinant host cell of the invention comprises any one ofthe polynucleotides or the recombinant vectors described therein.

A further recombinant cell host according to the invention comprises apolynucleotide containing a biallelic marker selected from the groupconsisting of A1 to A28, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith. Optionally, saidbiallelic marker is selected from the group consisting of A1 to A13,A15, A17 to A28, and the complements thereof, or optionally thebiallelic markers in linkage disequilibrium therewith

Preferred host cells used as recipients for the expression vectors ofthe invention are the following:

a) Prokaryotic host cells: Escherichia coli strains (I.E.DH5-α strain),Bacillus subtilis, Salmonella typhimurium, and strains from species likePseudomonas, Streptomyces and Staphylococcus.

b) Eukaryotic host cells: HeLa cells (ATCC No. CCL2; No. CCL2. 1; No.CCL2. 2), Cv 1 cells (ATCC No. CCL70), COS cells (ATCC No. CRL1650; No.CRL1651), Sf-9 cells (ATCC No. CRL1711), C127 cells (ATCC No. CRL-1804),3T3 (ATCC No. CRL-6361), CHO (ATCC No. CCL-61), human kidney 293. (ATCCNo. 45504; No. CRL-1573) and BHK (ECACC No. 84100501; No. 84111301).

c) Other mammalian host cells.

The FLAP gene expression in mammalian, and typically human, cells may berendered defective, or alternatively it may be proceeded with theinsertion of a FLAP genomic or cDNA sequence with the replacement of theFLAP gene counterpart in the genome of an animal cell by a FLAPpolynucleotide according to the invention. These genetic alterations maybe generated by homologous recombination events using specific DNAconstructs that have been previously described.

One kind of cell host that may be used are mammal zygotes, such asmurine zygotes. For example, murine zygotes may undergo microinjectionwith a purified DNA molecule of interest, for example a purified DNAmolecule that has previously been adjusted to a concentration range from1 ng/ml—for BAC inserts-3 ng/μl—for P1 bacteriophage inserts—in 10 mMTris-HCl, pH 7. 4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine,and 70 μM spermidine. When the DNA to be microinjected has a large size,polyamines and high salt concentrations can be used in order to avoidmechanical breakage of this DNA, as described by Schedl et al (1993b).

Anyone of the polynucleotides of the invention, including the DNAconstructs described herein, may be introduced in an embryonic stem (ES)cell line, preferably a mouse ES cell line. ES cell lines are derivedfrom pluripotent, uncommitted cells of the inner cell mass ofpre-implantation blastocysts. Preferred ES cell lines are the following:ES-E14TG2a (ATCC No. CRL-1821), ES-D3 (ATCC No. CRL1934 and No.CRL-11632), YS001 (ATCC No. CRL-11776), 36. 5 (ATCC No. CRL-11116). Tomaintain ES cells in an uncommitted state, they are cultured in thepresence of growth inhibited feeder cells which provide the appropriatesignals to preserve this embryonic phenotype and serve as a matrix forES cell adherence. Preferred feeder cells consist of primary embryonicfibroblasts that are established from tissue of day 13 to day 14 embryosof virtually any mouse strain, that are maintained in culture, such asdescribed by Abbondanzo et al. (1993) and are inhibited in growth byirradiation, such as described by Robertson (1987), or by the presenceof an inhibitory concentration of LIF, such as described by Pease andWilliams (1990).

The constructs in the host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.

Following transformation of a suitable host and growth of the host to anappropriate cell density, the selected promoter is induced byappropriate means, such as temperature shift or chemical induction, andcells are cultivated for an additional period.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means, and the resulting crude extract retained for furtherpurification.

Microbial cells employed in the expression of proteins can be disruptedby any convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known by the skill artisan.

Transgenic Animals

The terms “transgenic animals” or “host animals” are used hereindesignate animals that have their genome genetically and artificiallymanipulated so as to include one of the nucleic acids according to theinvention. Preferred animals are non-human mammals and include thosebelonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats)and Oryctogalus (e.g. rabbits) which have their genome artificially andgenetically altered by the insertion of a nucleic acid according to theinvention. In one embodiment, the invention encompasses non-human hostmammals and animals comprising a recombinant vector of the invention ora FLAP gene disrupted by homologous recombination with a knock outvector.

The transgenic animals of the invention all include within a pluralityof their cells a cloned recombinant or synthetic DNA sequence, morespecifically one of the purified or isolated nucleic acids comprising aFLAP coding sequence, a FLAP regulatory polynucleotide or a DNA sequenceencoding an antisense polynucleotide such as described in the presentspecification.

Generally, a transgenic animal according the present invention comprisesany one of the polynucleotides, the recombinant vectors and the cellhosts described in the present invention.

A further transgenic animal according to the invention contains in theirsomatic cells and/or in their germ line cells a polynucleotidecomprising a biallelic marker selected from the group consisting of A1to A28, and the complements thereof, or optionally the biallelic markersin linkage disequilibrium therewith. Optionally said biallelic marker isselected from the group consisting of A1 to A13, A15, A17 to A28, andthe complements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith.

In a first preferred embodiment, these transgenic animals may be goodexperimental models in order to study the diverse pathologies related tocell differentiation, in particular concerning the transgenic animalswithin the genome of which has been inserted one or several copies of apolynucleotide encoding a native FLAP protein, or alternatively a mutantFLAP protein.

In a second preferred embodiment, these transgenic animals may express adesired polypeptide of interest under the control of the regulatorypolynucleotides of the FLAP gene, leading to good yields in thesynthesis of this protein of interest, and eventually a tissue specificexpression of this protein of interest.

The design of the transgenic animals, including knock out animals, ofthe invention may be made according to the conventional techniques wellknown from the one skilled in the art. For more details regarding theproduction of transgenic animals, and specifically transgenic mice, itmay be referred to U.S. Pat. Nos. 4,873,191, issued Oct. 10, 1989,5,464,764 issued Nov. 7, 1995; 5,789,215, issued Aug. 4, 1998; Capecchi,M. R. (1989a); Capecchi, M. R. (1989b); and Tsuzuki, T. and Rancourt, D.E. (1998), these documents being hereby incorporated by reference.

The present invention encompasses knock out vectors comprising the novelpolynucleotides of the invention, as well as mammalian host cells andnon-human host mammals comprising a FLAP gene disrupted by homologousrecombination with such a knock out vector

Transgenic animals of the present invention are produced by theapplication of procedures which result in an animal with a genome thathas incorporated exogenous genetic material. The procedure involvesobtaining the genetic material, or a portion thereof, which encodeseither a FLAP coding sequence, a FLAP regulatory polynucleotide or a DNAsequence encoding a FLAP antisense polynucleotide such as described inthe present specification.

A recombinant polynucleotide of the invention is inserted into anembryonic or ES stem cell line. The insertion is preferably made usingelectroporation, such as described by Thomas et al. (1987). The cellssubjected to electroporation are screened (e.g. by selection viaselectable markers, by PCR or by Southern blot analysis) to findpositive cells which have integrated the exogenous recombinantpolynucleotide into their genome, preferably via an homologousrecombination event. An illustrative positive-negative selectionprocedure that may be used according to the invention is described byMansour et al. (1988).

Then, the positive cells are isolated, cloned and injected into 3.5 daysold blastocysts from mice, such as described by Bradley (1987). Theblastocysts are then inserted into a female host animal and allowed togrow to term.

Alternatively, the positive ES cells are brought into contact withembryos at the 2.5 days old 8-16 cell stage (morulae) such as describedby Wood et al. (1993) or by Nagy et al. (1993), the ES cells beinginternalized to colonize extensively the blastocyst including the cellswhich will give rise to the germ line.

The offspring of the female host are tested to determine which animalsare transgenic e.g. include the inserted exogenous DNA sequence andwhich are wild-type.

Thus, the present invention also concerns a transgenic animal containinga nucleic acid, a recombinant expression vector or a recombinant hostcell according to the invention.

Recombinant Cell Lines Derived from the Transgenic Animals of theInvention.

A further object of the invention consists of recombinant host cellsobtained from a transgenic animal described herein. In one embodimentthe invention encompasses cells derived from non-human host mammals andanimals comprising a recombinant vector of the invention or a FLAP genedisrupted by homologous recombination with a knock out vector.

Recombinant cell lines may be established in vitro from cells obtainedfrom any tissue of a transgenic animal according to the invention, forexample by transfection of primary cell cultures with vectors expressingone-genes such as SV40 large T antigen, as described by Chou (1989) andShay et al. (1991).

X-VII. Screening of Agents Acting on the Leukotriene Pathway

In a further embodiment, the present invention also concerns a methodfor the screening of new agents, or candidate substances, acting on theleukotriene pathway and which may be suitable for the treatment of apatient whose DNA comprises an allele of the FLAP gene associated with adisease involving the leukotriene pathway, more particularly asthma.

In a preferred embodiment, the invention relates to a method for thescreening of candidate substances for their ability to alter leukotrienebiosynthesis, preferably to identify active candidate substances withoutundesired side-effects such as increased liver transaminase levels. Themethod comprises the following steps: a) providing a cell line, anorgan, or a mammal expressing 5-LO and either a FLAP gene comprisingalleles for one or more FLAP-related biallelic markers, preferablyassociated with a modified leukotriene pathway, more preferably with adisease involving the leukotriene pathway such as asthma, or a mutatedFLAP gene comprising the trait cause mutation determined using theabove-noted method; b) obtaining a candidate substance; and, c) testingthe ability of the candidate substance to modify leukotrienebiosynthesis, and particularly to interact with the 5-LO and/or with theFLAP produced by the cell line or the transgenic mammal and/or to modifythe interaction between 5-LO and FLAP and/or to modulate the expressionlevels of FLAP.

In one embodiment of the above method, the method comprises providing acell line, an organ, or a mammal expressing 5-LO, a FLAP gene comprisingalleles for one or more FLAP-related biallelic markers, preferablyassociated with a modified leukotriene pathway, more preferably with adisease involving the leukotriene pathway such as asthma, and a mutatedFLAP gene comprising the trait cause mutation determined using theabove-noted method. Said biallelic markers may be selected from thegroup consisting of A1 to A28, and the complements thereof; Optionally,said FLAP-related biallelic marker may be selected from the groupconsisting of A1 to A13, A15, and A17 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, said biallelic markers are selected from thegroup consisting of A1 to A10 and A22 to A28, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, said biallelic markers are selected from thegroup consisting of A11 to A13, A15, A17 to A21, and the complementsthereof, or optionally the biallelic markers in linkage disequilibriumtherewith; optionally, said biallelic markers are selected from thegroup consisting of A14 or A16, and the complements thereof, oroptionally the biallelic markers in linkage disequilibrium therewith. Ina preferred embodiment, said biallelic markers are selected from thegroup consisting of A2, A14, A16, A18, A19, A22, and A23, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith. In another preferred embodiment said biallelicmarkers are selected from the group consisting of A14 and A19, and thecomplements thereof, or optionally the biallelic markers in linkagedisequilibrium therewith. In more preferred embodiment, said biallelicmarkers comprise the biallelic marker A19, and the complement thereof,or optionally the biallelic markers in linkage disequilibrium therewith.

A candidate substance is a substance which can interact with ormodulate, by binding or other intramolecular interactions. 5-LO or FLAP.Such substances may be potentially interesting for patients who are notresponsive to existing drugs. Screening may be effected using either invitro methods or in vivo methods.

In vitro methods can be carried out in numerous ways such as ontransformed cells which express the considered alleles of the FLAP genethrough 5-lipoxygenase activation and leukotriene synthesis measurementor on FLAP encoded by the considered allelic variant of FLAP throughFLAP binding assays.

Screening assays of the present invention generally involve determiningthe ability of a candidate substance to affect the activity of 5-LO orFLAP, such as the screening of candidate substances to identify thosethat inhibit or otherwise modify the function of 5-LO or FLAP in theleukotriene pathway.

One method of drug screening utilizes eukaryotic host cells which arestably transformed with recombinant polynucleotides expressing 5-LO andthe considered alleles of the FLAP gene. Such cells, either in viable orfixed form, can be used for standard binding assays. One can measure,for example, the formation of products of the leukotriene pathway suchas LTB₄ synthesis or examine the degree to which the formation of suchproducts is interfered with by the agent being tested.

Typically, this method includes preparing transformed cells whichexpress 5-LO and different forms of FLAP encoded by DNA sequencescontaining particular alleles of one or more of the biallelic markersand/or mutations described above. This is followed by testing the cellsexpressing the 5-LO and FLAP with a candidate substance to determine theability of the substance to affect the leukotriene pathway function, inorder to identify those which affect the enzymatic activity of 5-LO orthe activity of FLAP, and which thus can be suitable for use in humans.

Typical examples of such drug screening assays are provided below. It isto be understood that the parameters set forth in these examples can bemodified by the skilled person without undue experimentation.

Screening for 5-LO Inhibitors

Drug effects can be evaluated by assessing the 5-LO products generatedby cells expressing both the 5-LO gene and the considered allele of theFLAP gene. Eukaryotic cells previously transformed with appropriatevectors as described previously and expressing 5-LO and the allele ofthe FLAP gene under study are harvested by centrifugation (300 g, 5 min,and room temperature) and washed with an appropriate buffer. The cellsare then resuspended in buffer, pre-warmed at 37° C., preferably at acell density of 5×10⁶ cells/ml. Aliquots of the cell suspension areincubated with the considered drug for preferably 5 min at 37° C.Reaction is initiated by the addition of calcium ionophore A23187 andarachidonic acid. Following incubation at 37° C., reaction is stopped byadding methanol containing prostaglandin B2 as an internal standard forHPLC analysis. 5-LO reaction products are extracted into chloroform,dried under a stream of nitrogen, and resuspended in HPLC solvent. Thesamples are analyzed by reverse-phase HPLC using preferably an isocraticsolvent system of methanol/water/acetic acid (75:25:0.01). The elutionis monitored at preferably 270 and 234 nm. 5-LO products are quantitatedby comparison of peak areas to those of standard curves of authenticstandards, and corrected for minor differences in extraction efficiencydetermined using the prostaglandin B2 internal standard. This method isdescribed in more detail in Dixon et al. (1990) and Abramovitz et al.(1993), the disclosures of which are incorporated herein by reference.

Screening for FLAP Inhibitors

The FLAP protein or portions thereof described above may be used in drugscreening procedures to identify molecules which are agonists,antagonists, or inhibitors of FLAP activity. The FLAP protein or portionthereof used in such analyses may be free in solution or linked to asolid support. Alternatively, FLAP protein or portions thereof can beexpressed on a cell surface. The cell may naturally express the FLAPprotein or portion thereof or, Alternatively, the cell may express theFLAP protein or portion thereof from an expression vector such as thosedescribed above.

In one method of drug screening, eukaryotic or prokaryotic host cellswhich are stably transformed with recombinant polynucleotides in orderto express the FLAP protein or a portion thereof are used inconventional competitive binding assays or standard direct bindingassays. For example, the formation of a complex between the FLAP proteinor a portion thereof and the agent being tested may be measured indirect binding assays. Alternatively, the ability of a test agent toprevent formation of a complex between the FLAP protein or a portionthereof and a known ligand may be measured.

For example, a FLAP inhibitor binding assay can be based on theobservation that MK-886, an indole leukotriene biosynthesis inhibitor,binds with high affinity and specificity to FLAP. Binding of theconsidered drug to FLAP can be assessed by a competition experimentswith a radiolabeled analog of MK-886, ¹²⁵I-L-691-831. A suspension ofcells expressing the considered allele of FLAP containing preferably 210⁷ cells is centrifuged at 500×g for 10 min. The pelleted cells arethen resuspended in lysis buffer. This suspension is sonicated on ice bythree 20 see bursts. Cell lysis is checked visually. Binding isinitiated by addition of cell lysis samples to wells containing¹²⁵I-L-691-831 and either the considered drug or nothing (control). Theplate is incubated for 20 min at room temperature. The samples are thenfiltered and washed. Bound ¹²⁵I-L-691-831 is determined in a counter.Specific drug binding is defined as the difference between binding inthe absence and the presence of the considered drug. This FLAP bindingassay is described with more details in Charleson et al. (1992).

Alternatively, the high throughput screening techniques disclosed inpublished PCT application WO 84/03564 may be used. In such techniques,large numbers of small peptides to be tested for FLAP binding activityare synthesized on a surface and affixed thereto. The test peptides arecontacted with the FLAP protein or a portion thereof, followed by a washstep. The amount of FLAP protein or portion thereof which binds to thetest compound is quantitated using conventional techniques.

In some methods, FLAP protein or a portion thereof may be fixed to asurface and contacted with a test compound. After a washing step, theamount of test compound which binds to the FLAP protein or portionthereof is measured.

Screening for Inhibitors of the Interaction Between 5-LO and FLAP

Drug effects can be evaluated through the assessment of the interactionbetween 5-LO and FLAP proteins.

Interaction between 5-LO and FLAP protein may be assessed using twohybrid systems such as the Matchmaker Two Hybrid System 2 (Catalog NoK1604-1, Clontech). As described in the manual accompanying theMatchmaker Two Hybrid System 2 (Catalog No K1604-1, Clontech) nucleicacids encoding the FLAP protein or a portion thereof, are inserted intoan expression vector such that they are in frame with DNA encoding theDNA binding domain of the yeast transcriptional activator GAL4. 5-LOcDNA or a portion thereof is inserted into a second expression vectorsuch that they are in frame with DNA encoding the activation domain ofGAL4. The two expression plasmids are transformed into yeast and theyeast are plated on selection medium which selects for expression ofselectable markers on each of the expression vectors as well as GAL4dependent expression of the HIS3 gene. Transformants capable of growingon medium lacking histidine are screened for GAL4 dependent lacZexpression. Those cells which are positive in both the histidineselection and the lacZ assay contain interaction between FLAP and 5-LOproteins.

In another method, affinity columns containing the FLAP protein or aportion thereof can be constructed. In some versions of this method theaffinity columni contains chimeric proteins in which the FLAP protein ora portion thereof is fused to glutathione S-transferase. 5-LO protein isapplied to the affinity column. The 5-LO protein retained on theaffinity column can be measured and can allow assessment of theinteraction between FLAP and 5-LO proteins.

Association between 5-LO and FLAP proteins can also be assessed by usingan Optical Biosensor as described in Edwards et Leatherbarrow, (1997).The main advantage of the method is that it allows the determination ofthe association rate. Typically a FLAP molecule is linked to the sensorsurface (through a carboxymethyl dextran matrix) and a sample of 5-LOmolecules is placed in contact with the FLAP molecules. The binding of a5-LO molecule to the FLAP molecule causes a change in the refractiveindex and/or thickness. This change is detected by the Biosensorprovided it occurs in the evanescent field (which extend a few hundrednanometers from the sensor surface). Hence, the effect of candidate drugon the association between FLAP and 5-LO proteins can be easilymeasured.

Screening for Expression Modifiers

The screening of expression modifiers is important as it can be used fordetecting modifiers specific to one allele or a group of alleles of theFLAP gene. The alteration of FLAP expression in response to a modifiercan be determined by administering or combining the candidate modifierwith an expression system such as animal, or cell, and in in vitrotranscription assay.

The effect of the modifier on FLAP transcription and/or steady statemRNA levels can also be determined. As with the basic expressionlevels., tissue specific interactions are of interest. Correlations aremade between the ability of an expression modifier to affect FLAPactivity, and the presence of the targeted polymorphisms. A panel ofdifferent modifiers may be screened in order to determine the effectunder a number of different conditions.

Expression levels and patterns of FLAP may be analyzed by solutionhybridization with long probes as described in International PatentApplication No WO 97/05277, the entire contents of which areincorporated herein by reference. Briefly, the FLAP cDNA or the FLAPgenomic DNA described above, or fragments thereof, is inserted at acloning site immediately downstream of a bacteriophage (T3, T7 or SP6)RNA polymerase promoter to produce antisense RNA. Preferably, the FLAPinsert comprises at least 100 or more consecutive nucleotides of thegenomic DNA sequence or the cDNA sequences, particularly thosecomprising at least one of the biallelic markers of the presentinvention or those encoding mutated FLAP. The plasmid is linearized andtranscribed in the presence of ribonucleotides comprising modifiedribonucleotides (i.e. biotin-UTP and DIG-UTP). An excess of this doublylabeled RNA is hybridized in solution with mRNA isolated from cells ortissues of interest. The hybridizations are performed under standardstringent conditions (40-50° C. for 16 hours in an 80% formamide, 0.4 MNaCl buffer, pH 7-8). The unhybridized probe is removed by digestionwith ribonucleases specific for single-stranded RNA (i.e. RNases CL3,T1, Phy M, U2 or A). The presence of the biotin-UTP modification enablescapture of the hybrid on a microtitration plate coated withstreptavidin. The presence of the DIG modification enables the hybrid tobe detected and quantified by ELISA using an anti-DIG antibody coupledto alkaline phosphatase.

Quantitative analysis of FLAP gene expression may also be performedusing arrays. As used herein, the term array means a one dimensional,two dimensional, or multidimensional arrangement of a plurality ofnucleic acids of sufficient length to permit specific detection ofexpression of mRNAs capable of hybridizing thereto. For example, thearrays may contain a plurality of nucleic acids derived from genes whoseexpression levels are to be assessed. The arrays may include the FLAPgenomic DNA, the FLAP cDNA sequences or the sequences complementarythereto or fragments thereof, particularly those comprising at least oneof the biallelic markers of the present invention or those encodingmutated FLAP. Preferably, the fragments are at least 15 nucleotides inlength. In other embodiments, the fragments are at least 25 nucleotidesin length. In some embodiments, the fragments are at least 50nucleotides in length. More preferably, the fragments are at least 100nucleotides in length. In another preferred embodiment, the fragmentsare more than 100 nucleotides in length. In some embodiments thefragments may be more than 500 nucleotides in length.

For example, quantitative analysis of FLAP gene expression may beperformed with a complementary DNA microarray as described by Schena etal. (1995 and 1996). Full length FLAP cDNAs or fragments thereof areamplified by PCR and arrayed from a 96-well microtiter plate ontosilylated microscope slides using high-speed robotics. Printed arraysare incubated in a humid chamber to allow rehydration of the arrayelements and rinsed, once in 0.2% SDS for 1 min, twice in water for 1min and once for 5 min in sodium borohydride solution. The arrays aresubmerged in water for 2 min at 95° C., transferred into 0.2% SDS for 1min, rinsed twice with water, air dried and stored in the dark at 25° C.

Cell or tissue mRNA is isolated or commercially obtained and probes areprepared by a single round of reverse transcription. Probes arehybridized to 1 cm² microarrays under a 14×14 mm glass coverslip for6-12 hours at 60° C. Arrays are washed for 5 min at 25° C. in lowstringency wash buffer (1×SSC/0.2% SDS), then for 10 min at roomtemperature in high stringency wash buffer (0.1×SSC/0.2% SDS). Arraysare scanned in 0.1×SSC using a fluorescence laser scanning device fittedwith a custom filter set. Accurate differential expression measurementsare obtained by taking the average of the ratios of two independenthybridizations.

Quantitative analysis of FLAP gene expression may also be performed withfull length FLAP cDNAs or fragments thereof in complementary DNA arraysas described by Pietu et al. (1996). The full length FLAP cDNA orfragments thereof is PCR amplified and spotted on membranes. Then, mRNAsoriginating from various tissues or cells are labeled with radioactivenucleotides. After hybridization and washing in controlled conditions,the hybridized mRNAs are detected by phospho-imaging or autoradiography.Duplicate experiments are performed and a quantitative analysis ofdifferentially expressed mRNAs is then performed.

Alternatively, expression analysis using the FLAP genomic DNA, the FLAPcDNA, or fragments thereof can be done through high density nucleotidearrays as described by Lockhart et al. (1996) and Sosnowsky et al.(1997). Oligonucleotides of 15-50 nucleotides from the sequences of theFLAP genomic DNA, the FLAP cDNA sequences, particularly those comprisingat least one of the biallelic markers of the present invention or thoseencoding mutated FLAP, or the sequences complementary thereto, aresynthesized directly on the chip (Lockhart et al., 1996) or synthesizedand then addressed to the chip (Sosnowski et al., 1997). Preferably, theoligonucleotides are about 20 nucleotides in length.

FLAP cDNA probes labeled with an appropriate compound, such as biotin,digoxigenin or fluorescent dye, are synthesized from the appropriatemRNA population and then randomly fragmented to an average size of 50 to100 nucleotides. The said probes are then hybridized to the chip. Afterwashing as described in Lockhart et al., supra and application ofdifferent electric fields (Sosnowsky et al., 1997)., the dyes orlabeling compounds are detected and quantified. Duplicate hybridizationsare performed. Comparative analysis of the intensity of the signaloriginating from cDNA probes on the same target oligonucleotide indifferent cDNA samples indicates a differential expression of FLAP mRNA.

Screening Using Transgenic Animals

In vivo methods can utilize transgenic animals for drug screening.Nucleic acids including at least one of the biallelic polymorphisms ofinterest can be used to generate genetically modified non-human animalsor to generate site specific gene modifications in cell lines. The term“transgenic” is intended to encompass genetically modified animalshaving a deletion or other knock-out of FLAP gene activity, having anexogenous FLAP gene that is stably transmitted in the host cells, orhaving an exogenous FLAP promoter operably linked to a reporter gene.Transgenic animals may be made through homologous recombination, wherethe FLAP locus is altered. Alternatively a nucleic acid construct israndomly integrated into the genome. Vectors for stable integrationinclude for example plasmids, retroviruses and other animal viruses, andYACs. Of interest are transgenic mammals e.g. cows, pigs, goats, horses,and particularly rodents such as rats and mice. Transgenic animals allowto study both efficacy and toxicity of the candidate drug.

XVIII. Computer-Related Embodiments

As used herein the term “nucleic acid codes of the invention” encompassthe nucleotide sequences comprising, consisting essentially of, orconsisting of any one of the following: a) a contiguous span of at least12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500,or 1000 nucleotides of SEQ ID NO: 1, wherein said contiguous spancomprises at least 1 of the following nucleotide positions of SEQ ID NO:1:1-7007, 8117-15994, 16550-24058, 24598-27872, 28413-35976, and36927-43069; b) a contiguous span of at least 12, 15, 18, 20, 25, 30,35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides ofSEQ ID NO: 1, wherein said contiguous span comprises a C at position16348 of SEQ ID NO: 1; c) a contiguous span of at least 12, 15, 18, 20,25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000nucleotides of SEQ ID NO: 1, wherein said contiguous span comprises thefollowing nucleotide positions of SEQ ID NO: 1: 7612-7637, 24060-24061,24067-24068, 27903-27905, and 28327-28329; d) a contiguous span of atleast 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,500, or 1000 nucleotides of SEQ ID NO: 1, wherein said contiguous spancomprises a nucleotide selected from the group consisting of an A atposition 7445, an A at position 7870, a T at position 16288, an A atposition 16383, a T at position 24361, a G at position 28336, a T atposition 28368, an A at position 36183, and a G at position 36509 of SEQID NO: 1; e) a contiguous span of at least 12, 15, 18, 20, 25, 30, 35,40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQID NO: 2, wherein said contiguous span comprises a T at position 197, anA at position 453, or a G at position 779 of SEQ ID NO: 2; and f) anucleotide sequence complementary to any one of the preceding nucleotidesequences.

The “nucleic acid codes of the invention” further encompass nucleotidesequences homologous to a contiguous span of at least 30, 35, 40, 50,60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of the followingnucleotide position range: 1-7007, 8117-15994, 16550-24058, 24598-27872,28413-35976, and 36927-43069 of SEQ ID NO: 1, and sequencescomplementary to all of the preceding sequences. Homologous sequencesrefer to a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%,80%, or 75% homology to these contiguous spans. Homology may bedetermined using any method described herein, including BLAST2N with thedefault parameters or with any modified parameters. Homologous sequencesalso may include RNA sequences in which uridines replace the thymines inthe nucleic acid codes of the invention. It will be appreciated that thenucleic acid codes of the invention can be represented in thetraditional single character format (See the inside back cover ofStryer, Lubert. Biochemistry, 3^(rd) edition. W. H Freeman & Co., NewYork.) or in any other format or code which records the identity of thenucleotides in a sequence.

As used herein the term “polypeptide codes of the invention” encompassthe polypeptide sequences comprising a contiguous span of at least 6, 8,10, 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID NO: 3,wherein said contiguous span includes an isoleucine residue at aminoacid position 127 of SEQ ID NO: 3. It will be appreciated that thepolypeptide codes of the invention can be represented in the traditionalsingle character format or three letter format (See the inside backcover of Stryer, Lubert. Biochemistry, 3^(rd) edition. W. H Freeman &Co., New York.) or in any other format or code which records theidentity of the polypeptides in a sequence.

It will be appreciated by those skilled in the art that the nucleic acidcodes of the invention and polypeptide codes of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer. As used herein, the words “recorded” and“stored” refer to a process for storing information on a computermedium. A skilled artisan can readily adopt any of the presently knownmethods for recording information on a computer readable medium togenerate manufactures comprising one or more of the nucleic acid codesof the invention, or one or more of the polypeptide codes of theinvention. Another aspect of the present invention is a computerreadable medium having recorded thereon at least 2, 5, 10, 15, 20, 25,30, or 50 nucleic acid codes of the invention. Another aspect of thepresent invention is a computer readable medium having recorded thereonat least 2, 5, 10, 15, 20, 25, 30, or 50 polypeptide codes of theinvention.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disc, afloppy disc, a magnetic tape, CD-ROM, DVD, RAM., or ROM as well as othertypes of other media known to those skilled in the art.

Embodiments of the present invention include systems, particularlycomputer systems which contain the sequence information describedherein. As used herein, “a computer system” refers to the hardwarecomponents, software components, and data storage components used tostore and/or analyze the nucleotide sequences of the nucleic acid codesof the invention, the amino acid sequences of the polypeptide codes ofthe invention, or other sequences. The computer system preferablyincludes the computer readable media described above, and a processorfor accessing and manipulating the sequence data.

Preferably, the computer is a general purpose system that comprises acentral processing unit (CPU), one or more data storage components forstoring data, and one or more data retrieving devices for retrieving thedata stored on the data storage components. A skilled artisan canreadily appreciate that any one of the currently available computersystems are suitable.

In one particular embodiment, the computer system includes a processorconnected to a bus which is connected to a main memory, preferablyimplemented as RAM, and one or more data storage devices, such as a harddrive and/or other computer readable media having data recorded thereon.In some embodiments, the computer system further includes one or moredata retrieving devices for reading the data stored on the data storagecomponents. The data retrieving device may represent, for example, afloppy disk drive, a compact disk drive, a magnetic tape drive, a harddisk drive, a CD-ROM drive, a DVD drive, etc. In some embodiments, thedata storage component is a removable computer readable medium such as afloppy disk, a compact disk, a magnetic tape, etc. containing controllogic and/or data recorded thereon. The computer system mayadvantageously include or be programmed by appropriate software forreading the control logic and/or the data from the data storagecomponent once inserted in the data retrieving device. Software foraccessing and processing the nucleotide sequences of the nucleic acidcodes of the invention, or the amino acid sequences of the polypeptidecodes of the invention (such as search tools, compare tools, modelingtools, etc.) may reside in main memory during execution.

In some embodiments, the computer system may further comprise a sequencecomparer for comparing the nucleic acid codes of the invention orpolypeptide codes of the invention stored on a computer readable mediumto reference nucleotide or polypeptide sequences stored on a computerreadable medium. A “sequence comparer” refers to one or more programswhich are implemented on the computer system to compare a nucleotide orpolypeptide sequence with other nucleotide or polypeptide sequencesand/or compounds including but not limited to peptides, peptidomimetics,and chemicals the sequences or structures of which are stored within thedata storage means. For example, the sequence comparer may compare thenucleotide sequences of the nucleic acid codes of the invention, or theamino acid sequences of the polypeptide codes of the invention stored ona computer readable medium to reference sequences stored on a computerreadable medium to identify homologies, motifs implicated in biologicalfunction, or structural motifs. The various sequence comparer programsidentified elsewhere in this patent specification are particularlycontemplated for use in this aspect of the invention.

Accordingly, one aspect of the present invention is a computer systemcomprising a processor, a data storage device having stored thereon anucleic acid code of the invention or a polypeptide code of theinvention, a data storage device having retrievably stored thereonreference nucleotide sequences or polypeptide sequences to be comparedto the nucleic acid code of the invention or polypeptide code of theinvention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify structural motifs in the nucleic acid code of theinvention and polypeptide codes of the invention or it may identifystructural motifs in sequences which are compared to these nucleic acidcodes and polypeptide codes. In some embodiments, the data storagedevice may have stored thereon the sequences of at least 2, 5, 10, 15,20, 25, 30, or 50 of the nucleic acid codes of the invention orpolypeptide codes of the invention.

Another aspect of the present invention is a method for determining thelevel of homology between a nucleic acid code of the invention and areference nucleotide sequence, comprising the steps of reading thenucleic acid code and the reference nucleotide sequence through the useof a computer program which determines homology levels and determininghomology between the nucleic acid code and the reference nucleotidesequence with the computer program. The computer program may be any of anumber of computer programs for determining homology levels, includingthose specifically enumerated herein, including BLAST2N with the defaultparameters or with any modified parameters. The method may beimplemented using the computer systems described above. The method mayalso be performed by reading 2, 5, 10, 15, 20, 25, 30, or 50 of theabove described nucleic acid codes of the invention through the use ofthe computer program and determining homology between the nucleic acidcodes and reference nucleotide sequences.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of the nucleic acid codes of thepresent invention, to reference nucleotide sequences in order todetermine whether the nucleic acid code of the invention differs from areference nucleic acid sequence at one or more positions. Optionallysuch a program records the length and identity of inserted, deleted orsubstituted nucleotides with respect to the sequence of either thereference polynucleotide or the nucleic acid code of the invention. Inone embodiment, the computer program may be a program which determineswhether the nucleotide sequences of the nucleic acid codes of theinvention contain one or more single nucleotide polymorphisms (SNP) withrespect to a reference nucleotide sequence. These single nucleotidepolymorphisms may each comprise a single base substitution, insertion,or deletion.

Another aspect of the present invention is a method for determining thelevel of homology between a polypeptide code of the invention and areference polypeptide sequence, comprising the steps of reading thepolypeptide code of the invention and the reference polypeptide sequencethrough use of a computer program which determines homology levels anddetermining homology between the polypeptide code and the referencepolypeptide sequence using the computer program.

Accordingly, another aspect of the present invention is a method fordetermining whether a nucleic acid code of the invention differs at oneor more nucleotides from a reference nucleotide sequence comprising thesteps of reading the nucleic acid code and the reference nucleotidesequence through use of a computer program which identifies differencesbetween nucleic acid sequences and identifying differences between thenucleic acid code and the reference nucleotide sequence with thecomputer program. In some embodiments, the computer program is a programwhich identifies single nucleotide polymorphisms. The method may beimplemented by the computer systems described above. The method may alsobe performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 50 of thenucleic acid codes of the invention and the reference nucleotidesequences through the use of the computer program and identifyingdifferences between the nucleic acid codes and the reference nucleotidesequences with the computer program.

In other embodiments the computer based system may further comprise anidentifier for identifying features within the nucleotide sequences ofthe nucleic acid codes of the invention or the amino acid sequences ofthe polypeptide codes of the invention.

An “identifier” refers to one or more programs which identifies certainfeatures within the above-described nucleotide sequences of the nucleicacid codes of the invention or the amino acid sequences of thepolypeptide codes of the invention.

The nucleic acid codes of the invention or the polypeptide codes of theinvention may be stored and manipulated in a variety of data processorprograms in a variety of formats. For example, they may be stored astext in a word processing file, such as MicrosoftWORD or WORDPERFECT oras an ASC11 file in a variety of database programs familiar to those ofskill in the art, such as DB2, SYBASE, or ORACLE. In addition, manycomputer programs and databases may be used as sequence comparers,identifiers, or sources of reference nucleotide or polypeptide sequencesto be compared to the nucleic acid codes of the invention or thepolypeptide codes of the invention. The following list is intended notto limit the invention but to provide guidance to programs and databaseswhich are useful with the nucleic acid codes of the invention or thepolypeptide codes of the invention. The programs and databases which maybe used include, but are not limited to: MacPattern (EMBL),DiscoveryBase (Molecular Applications Group), GeneMine (MolecularApplications Group), Look (Molecular Applications Group), MacLook(Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN andBLASTX (Altschul et al, 1990), FASTA (Pearson and Lipman, 1988), FASTDB(Brutlag et al., 1990), Catalyst (Molecular Simulations Inc.),Catalyst/SHAPE (Molecular Simulations Inc.), Cerius².DBAccess (MolecularSimulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II,(Molecular Simulations Inc.), Discover (Molecular Simulations Inc.),CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.),DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular SimulationsInc.), Homology (Molecular Simulations Inc.), Modeler (MolecularSimulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/ProteinDesign (Molecular Simulations Inc.), WebLab (Molecular SimulationsInc.), WebLab Diversity Explorer (Molecular Simulations Inc.), GeneExplorer Molecular Simulations Inc.), SeqFold (Molecular SimulationsInc.), the EMBL/Swissprotein database, the MDL Available ChemicalsDirectory database, the MDL Drug Data Report data base, theComprehensive Medicinal Chemistry database, Derwents's World Drug Indexdatabase, the BioByteMasterFile database, the Genbank database, and theGenseqn database. Many other programs and data bases would be apparentto one of skill in the art given the present disclosure.

Throughout this application, various publications, patents, andpublished patent applications are cited. The disclosures of thepublications, patents, and published patent specifications referenced inthis application are hereby incorporated by reference into the presentdisclosure to more fully describe the state of the art to which thisinvention pertains.

EXAMPLES Example 1 Detection of FLAP Biallelic Markers DNA Extraction

Donors were unrelated and healthy. They presented a sufficient diversityfor being representative of a French heterogeneous population. The DNAfrom 100 individuals was extracted and tested for the detection of thebiallelic markers.

30 ml of peripheral venous blood were taken from each donor in thepresence of EDTA. Cells (pellet) were collected after centrifugation for10 minutes at 2000 rpm. Red cells were lysed by a lysis solution (50 mlfinal volume: 10 mM Tris pH7.6; 5 mM MgCl₂; 10 mM NaCl). The solutionwas centrifuged (10 minutes, 2000 rpm) as many times as necessary toeliminate the residual red cells present in the supernatant, afterresuspension of the pellet in the lysis solution.

The pellet of white cells was lysed overnight at 42° C. with 3.7 ml oflysis solution composed of:

-   -   3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M;    -   200 μl SDS 10%; and    -   500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M).

For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) wasadded. After vigorous agitation, the solution was centrifuged for 20minutes at 10000 rpm.

For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were addedto the previous supernatant, and the solution was centrifuged for 30minutes at 2000 rpm. The DNA solution was rinsed three times with 70%ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm.The pellet was dried at 37° C., and resuspended in 1 ml TE 10-1 or 1 mlwater. The DNA concentration was evaluated by measuring the OD at 260 nm(1 unit OD=50 μg/ml DNA).

To determine the presence of proteins in the DNA solution, the OD 260/OD280 ratio was determined. Only DNA preparations having a OD 260/OD 280ratio between 1.8 and 2 were used in the subsequent examples describedbelow.

The pool was constituted by mixing equivalent quantities of DNA fromeach individual.

Example 2 Detection of the Biallelic Markers Amplification of GenomicDNA by PCR

The amplification of specific genomic sequences of the DNA samples ofexample 1 was carried out on the pool of DNA obtained previously. Inaddition, 50 individual samples were similarly amplified.

PCR assays were performed using the following protocol:

Final volume 25 μl DNA 2 ng/μl MgCl₂ 2 mM dNTP (each) 200 μM primer(each) 2.9 ng/μl Ampli Taq Gold DNA polymerase 0.05 unit/μl PCR buffer(10x = 0.1 M Tris-HCl pH 8.3 0.5M KCl) 1x

Each pair of first primers was designed using the sequence informationof the FLAP gene (GenBank 182657, Kennedy et al. 1991 incorporatedherein by reference) and the OSP software (Hillier & Green, 1991). Thesefirst primers had about 20 nucleotides in length and their respectivesequences are disclosed in Table 1.

TABLE 1 Complementary Position range of the Position range of positionrange of amplicon in SEQ ID amplification primer amplification primerAmplicon NO: 1 PU in SEQ ID NO: 1 RP in SEQ ID NO: 1 10-517 3851 4189B15 3851 3869 C15 4171 4189 10-518 4120 4390 B16 4120 4138 C16 4372 439010-253 4373 4792 B1 4373 4391 C1 4773 4792 10-499 4814 5043 B2 4814 4833C2 5026 5043 10-500 4956 5422 B3 4956 4972 C3 5405 5422 10-522 5524 5996B17 5524 5542 C17 5978 5996 10-503 6218 6672 B4 6218 6235 C4 6652 667210-504 6522 6790 B5 6522 6539 C5 6772 6790 10-204 7120 7574 B6 7120 7137C6 7557 7574 10-32 7513 7933 B7 7513 7531 C7 7914 7933 10-33 16114 16533B8 16114 16132 C8 16515 16533 10-34 24072 24425 B9 24072 24089 C9 2440824425 10-35 27978 28401 B10 27978 27995 C10 28384 28401 10-36 3602036465 B11 36020 36039 C11 36446 36465 10-498 36318 36669 B12 36318 36337C12 36652 36669 12-629 38441 38840 B13 38441 38460 C13 38820 3884012-628 42233 42749 B14 42233 42253 C14 42731 42749

Preferably, the primers contained a common oligonucleotide tail upstreamof the specific bases targeted for amplification which was useful forsequencing.

PU Amplification Primers contain the following additional PU 5′sequence: TGTAAAACGACGGCCAGT (SEQ ID NO: 14); RP amplification primerscontain the following RP 5′ sequence: CAGGAAACAGCTATGACC (SEQ ID NO:15).

The synthesis of these primers was performed following thephosphoramidite method, on a GENSET UFPS 24.1 synthesizer.

DNA amplification was performed on a Genius II thermocycler. Afterheating at 94° C. for 10 min, 40 cycles were performed. Each cyclecomprised: 30 sec at 94° C., 55° C. for 1 min, and 30 sec at 72° C. Forfinal elongation, 7 min at 72° C. end the amplification. The quantitiesof the amplification products obtained were determined on 96-wellmicrotiter plates, using a fluorometer and Picogreen as intercalantagent (Molecular Probes).

Example 3 Detection of the Biallelic Markers Sequencing of AmplifiedGenomic DNA and Identification of Polymorphisms

The sequencing of the amplified DNA obtained in example 2 was carriedout on ABI 377 sequencers. The sequences of the amplification productswere determined using automated dideoxy terminator sequencing reactionswith a dye terminator cycle sequencing protocol. The products of thesequencing reactions were run on sequencing gels and the sequences weredetermined.

The sequence data were further evaluated for polymorphisms by detectingthe presence of biallelic markers among the pooled amplified fragments.The polymorphism search was based on the presence of superimposed peaksin the electrophoresis pattern resulting from different bases occurringat the same position.

17 fragments of amplification were analyzed. In these segments, 28biallelic markers were detected. The localization of the biallelicmarkers was as shown in Table 2.

TABLE 2 Freq. Polymor- BM position Position of 47 Ampli- Marker ofLocalization phism in SEQ ID 47mers in mers con BM Name all2 in FLAPgene all1 all2 No 1 No 2 SEQ ID NO: name 10-517 A25 10-517-1005′regulatory G C 3950 3927 3973 P25 10-518 A26 10-518-125 5′regulatory GT 4243 4220 4266 P26 10-518 A27 10-518-194 5′regulatory A G 4312 42894335 P27 10-253 A1 10-253-118 5′regulatory A G 4490 4467 4513 P1 10-253A2 10-253-298 4.57 5′regulatory G C 4670 4647 4693 P2 10-253 A310-253-315 5′regulatory C T 4687 4664 4710 P3 10-499 A4 10-499-1555′regulatory A G 4968 4945 4991 P4 10-500 A5 10-500-185 5′regulatory C T5140 5117 5163 P5 10-500 A6 10-500-258 5′regulatory G T 5213 5190 5236P6 10-500 A7 10-500-410 5′regulatory A G 5364 5341 5387 P7 10-522 A2810-522-71 5′regulatory A G 5594 5571 5617 P28 10-503 A8 10-503-1595′regulatory G T 6370 6347 6393 P8 10-504 A9 10-504-172 5′regulatory A T6693 6670 6716 P9 10-504 A10 10-504-243 5′regulatory A C 6763 6740 6786P10 10-204 A11 10-204-326 6.63 5′regulatory A G 7445 7422 7468 P11 10-32A12 10-32-357 33.45 Intron 1 A C 7870 7847 7893 P12 10-33 A13 10-33-1752.3 Exon 2 C T 16288 197 16265 16311 P13 10-33 A14 10-33-234 43.98Intron 2 A C 16347 16324 16370 P14 10-33 A15 10-33-270 Intron 2 A G16383 16360 16406 P15 10-33 A16 10-33-327 24.26 Intron 2 C T 16440 1641716463 P16 10-34 A17 10-34-290 Intron 3 G T 24361 24338 24384 P17 10-35A18 10-35-358 31.25 Intron 4 G C 28336 28313 28359 P18 10-35 A1910-35-390 22.98 Intron 4 C T 28368 28345 28391 P19 10-36 A20 10-36-164Exon 5 A G 36183 453 36160 36206 P20 V127→I 10-498 A21 10-498-192 Exon 5A G 36509 779 36486 36532 P21 12-629 A22 12-629-241 28.3 3′regulatory GC 38681 38658 38704 P22 12-628 A24 12-628-311 3′regulatory T C 4244042417 42463 P24 12-628 A23 12-628-306 10.27 3′regulatory G A 42445 4242242468 P23

BM refers to “biallelic marker”. All1 and all2 refer respectively toallele 1 and allele 2 of the biallelic marker. “Freq. Of all2” refers tothe frequency of the allele 2 in percentage in Caucasian US controlpopulation, except for the biallelic marker 10-204/326 for which thepopulation is the French Caucasian controls. Frequencies corresponded toa population of random blood donors from French Caucasian origin.

The polymorphisms A14 (10-33-234) and A16 (10-33-327) have been observedin Kennedy et al, 1991. However, their frequencies in the population wasunknown, therefore they can not be considered validated biallelicmarkers, until the results of the present inventors were obtained.

Example 4 Validation of the Polymorphisms Through Microsequencing

The biallelic markers identified in example 3 were further confirmed andtheir respective frequencies were determined through microsequencing.Microsequencing was carried out for each individual DNA sample describedin Example 1.

Amplification from genomic DNA of individuals was performed by PCR asdescribed above for the detection of the biallelic markers with the sameset of PCR primers (Table 1).

The preferred primers used in microsequencing had about 19 nucleotidesin length and hybridized just upstream of the considered polymorphicbase. Their sequences are disclosed in Table 3 below.

TABLE 3 Position range of Complementary position microsequencing rangeof Biallelic primer mis 1 in microsequencing primer Marker Name MarkerMis. 1 SEQ ID NO: 1 Mis. 2 mis. 2 in SEQ ID NO: 1 10-517-100 A25 D253930 3949 E25 3951 3970 10-518-125 A26 D26 4223 4242 E26 4244 426310-518-194 A27 D27 4292 4311 E27 4313 4332 10-253-118 A1 D1 4470 4489 E14491 4510 10-253-298 A2 D2 4650 4669 E2 4671 4690 10-253-315 A3 D3 46674686 E3 4688 4707 10-499-155 A4 D4 4948 4967 E4 4969 4988 10-500-185 A5D5 5120 5139 E5 5141 5160 10-500-258 A6 D6 5193 5212 E6 5214 523310-500-410 A7 D7 5344 5363 E7 5365 5384 10-522-71 A28 D28 5574 5593 E285595 5614 10-503-159 A8 D8 6350 6369 E8 6371 6390 10-504-172 A9 D9 66736692 E9 6694 6713 10-504-243 A10 D10 6743 6762 E10 6764 6783 10-204-326A11 D11 7425 7444 E11 7446 7465 10-32-357 A12 D12 7850 7869 E12 78717890 10-33-175 A13 D13 16268 16287 E13 16289 16308 10-33-234 A14 D1416327 16346 E14 16348 16367 10-33-270 A15 D15 16363 16382 E15 1638416403 10-33-327 A16 D16 16420 16439 E16 16441 16460 10-34-290 A17 D1724341 24360 E17 24362 24381 10-35-358 A18 D18 28316 28335 E18 2833728356 10-35-390 A19 D19 28348 28367 E19 28369 28388 10-36-164 A20 D2036163 36182 E20 36184 36203 10-498-192 A21 D21 36489 36508 E21 3651036529 12-629-241 A22 D22 38661 38680 E22 38682 38701 12-628-311 A24 D2442420 42439 E24 42441 42460 12-628-306 A23 D23 42425 42444 E23 4244642465

M is 1 and M is 2 respectively refer to microsequencing primers whichhybridized with the non-coding strand of the FLAP gene or with thecoding strand of the FLAP gene.

The microsequencing reaction was performed as follows:

5 μl of PCR products were added to 5 μl purification mix 2U SAP (Shrimpalkaline phosphate) (Amersham E70092X)); 2U Exonuclease I (AmershamE70073Z); 1 μl SAP buffer (200 mM Tris-HCl pH8, 100 mM MgCl₂) in amicrotiter plate. The reaction mixture was incubated 30 minutes at 37°C., and denatured 10 minutes at 94° C. afterwards. To each well was thenadded 20 μl of microsequencing reaction mixture containing: 10 μmolmicrosequencing oligonucleotide (19mers, GENSET, crude synthesis, 50D),1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer(260 mM Tris HCl pH 9.5, 65 nM MgCl₂), and the two appropriatefluorescent ddNTPs complementary to the nucleotides at the polymorphicsite corresponding to both polymorphic bases (11.25 nM TAMRA-ddTTP;16.25 nM ROX-ddCTP; 1.675 nM REG-ddATP; 1.25 μM RHO-ddGTP; Perkin Elmer,Dye Terminator Set 401095). After 4 minutes at 94° C., 20 PCR cycles of15 sec at 55° C., 5 sec at 72° C., and 10 see at 94° C. were carried outin a Tetrad PTC-225 thermocycler (MJ Research). The microtiter plate wascentrifuged 10 sec at 1500 rpm. The unincorporated dye terminators wereremoved by precipitation with 19 μl MgCl₂ mM and 55 μl 100% ethanol.After 15 minute incubation at room temperature. The microtiter plate wascentrifuged at 3300 rpm 15 minutes at 4° C. After discarding thesupernatants, the microplate was evaporated to dryness under reducedpressure (Speed Vac); samples were resuspended in 2.5 μl formamide EDTAloading buffer and heated for 2 min at 95° C. 0.8 μl microsequencingreaction were loaded on a 10% (19:1) polyacrylamide sequencing gel. Thedata were collected by an ABI PRISM 377 DNA sequencer and processedusing the GENESCAN software (Perkin Elmer).

Example 5 Association Study Between Asthma and the Biallelic Markers ofthe Flap Gene Collection of DNA Samples from Affected and Non-AffectedIndividuals

The disease trait followed in this association study was asthma, adisease involving the leukotriene pathway.

The asthmatic population corresponded to 297 individuals that took partin a clinical study for the evaluation of the anti-asthmatic drugZileuton. More than 90% of these 297 asthmatic individuals had aCaucasian ethnic background.

The control population corresponded to unaffected individuals. In thisassociation study, either Caucasian French population (190 individuals)or Caucasian US population (286 individuals) is used as controlpopulation. The preferred control population is the Caucasian USpopulation since the asthmatic population essentially comprises USindividuals.

Example 6 Association Study Between Asthma and the Biallelic Markers ofthe FLAP Gene Genotyping of Affected and Control Individuals

The general strategy to perform the association studies was toindividually scan the DNA samples from all individuals in each of thepopulations described above in order to establish the allele frequenciesof the above described biallelic markers in each of these populations.

Allelic frequencies of the above-described biallelic markers in eachpopulation were determined by performing microsequencing reactions onamplified fragments obtained by genomic PCR performed on the DNA samplesfrom each individual. Genomic PCR and microsequencing were performed asdetailed above in examples 2 and 4 using the described PCR andmicrosequencing primers.

Example 7 Association Study Between Asthma and the Biallelic Markers ofthe FLAP Gene

A) Association Studies for Asthma Gene with Caucasian French ControlPopulation

This association study uses 293 asthmatic individuals and 185 CaucasianFrench controls.

As shown in FIG. 2 (A), markers 10-32/357 and 10-35/390 presented astrong association with asthma, this association being highlysignificant (pvalue=1.95×10⁻³ for marker 10-32/357 and 1.75×10⁻³ formarker 10-35-390). The two markers 10-32/357 and 10-35/390 can be thenused in diagnostics with a test based on each marker. Two other markersshowed moderate association when tested independently, namely 33/234,and 35/358.

B) Association Studies for Asthma Gene with Caucasian US ControlPopulation

This association study uses 297 asthmatic individuals and 286 CaucasianUS controls.

As shown in FIG. 2 (B), the biallelic marker 10-35/390 presented astrong association with asthma, this association being highlysignificant (pvalue=2.29×10⁻³). The two markers 10-32/357 and 10-33/234showed weak association when tested independently.

The biallelic marker 10-35/390 is located in the genomic sequence ofFLAP. Therefore, the association studies results show that apolymorphism of the FLAP gene seems to be related to asthma. Thebiallelic marker 10-35/390 can be then used in diagnostics with a testbased on this marker or on a combination of biallelic markers comprisingthis marker.

Example 8 Association Studies Haplotype Frequency Analysis

One way of increasing the statistical power of individual markers, is byperforming haplotype association analysis.

Haplotype analysis for association of FLAP markers and asthma wasperformed by estimating the frequencies of all possible haplotypescomprising biallelic markers selected from the group consisting of10-253/298, 10-32/357, 10-33/175, 10-33/234, 10-33/327, 10-35/358,10-35/390, 12-628/306, and 12-629/241 in the asthmatic and Caucasian UScontrol populations described in Example 7, and comparing thesefrequencies by means of a chi square statistical test (one degree offreedom). Haplotype estimations were performed by applying theExpectation-Maximization (EM) algorithm (Excoffier L & Slatkin M, 1995),using the EM-HAPLO program (Hawley M E, Pakstis A J & Kidd K K, 1994).

The most significant haplotypes obtained are shown in FIG. 3.

The preferred two-markers haplotypes, described in FIG. 3 as HAP1 toHAP7, comprise either the marker 10-33/234 (allele A) or the marker10-35/390 (allele T). The more preferred two-markers haplotype HAP1 (Aat 10-33/234 and T at 10-35/390) presented a p-value of 8.2×10⁻⁴ and anodd-ratio of 1.61. Estimated haplotype frequencies were 28.3% in thecases and 19.7% in the US controls. Two other two-haplotypes HAP2 (A at10-33/234 and G at 12-629/241) and HAP3 (T at 10-33/327 and T at10-33/390) presented respectively a p-value of 1.6×10⁻³ and 1.8×10⁻³, anodd-ratio of 1.65 and 1.53 and haplotypes frequencies of 0.305 and 0.307for asthmatic population and of 0.210 and 0.224 for US controlpopulation.

Preferred three-markers haplotypes comprise the marker 10-33/234 (alleleA) and the marker 10-35/390 (allele T): HAP37, HAP38, HAP39 and HAP41.The more preferred three-markers haplotype HAP37 (A at 10-33/234, T at10-33/390 and C at 12-628/306) presented a p-value of 8.6×10⁻⁴ and anodd-ratio of 1.76. Estimated haplotype frequencies were 26.5% in thecases and 17.1% in the US controls. A further three-markers haplotypeHAP40 (A at 10-33/234, C at 12-628/306 and G at 12-629/241) is alsosignificant.

Four-markers haplotypes (HAP121 to HAP125), five-markers haplotypes (P247 and 248) and a six-markers haplotype (HAP373) showed significantp-values. They all comprise the marker 10-33/234 (allele A) and themarker 10-35/390 (allele T), except the haplotype HAP124 which does notcomprise the marker 10-35/390. The other markers are chosen from thegroup consisting of 10-235/298 (allele C), 10-35/358 (allele G),12-628/306 (allele C) and 12-629/241 (allele G).

The more preferred haplotype comprising A at 10-33/234 and T at10-35/390 (HAP1 in FIG. 3) is also significant in a haplotype frequencyanalysis with asthmatic population and Caucasian French controls.Indeed, this haplotype presented a p-value of 2.7×10⁻³ and an odd-ratioof 1.67. Estimated haplotype frequencies were 28.3% in the cases and19.2% in the French controls (see FIG. 4).

The haplotype HAP1 is the more preferred haplotype of the invention. Itcan be used in diagnosis of asthma. Moreover, most of the significanthaplotypes associated with asthma comprise the biallelic marker10-35/390 (allele A) and could also be used in diagnosis.

The statistical significance of the results obtained for the haplotypeanalysis was evaluated by a phenotypic permutation test reiterated 1000or 10,000 times on a computer. For this computer simulation, data fromthe asthmatic and control individuals were pooled and randomly allocatedto two groups which contained the same number of individuals as thecase-control populations used to produce the data summarized in FIG. 3.A haplotype analysis was then run on these artificial groups for the 2markers included in the haplotype HAP1 which, showed the strongestassociation with asthma. This experiment was reiterated 1000 and 10,000times and the results are shown in FIG. 4. These results demonstratethat among 1000 iterations none and among 10,000 iterations only 1 ofthe obtained haplotypes had a p-value comparable to the one obtained forthe haplotype HAP1. These results clearly validate the statisticalsignificance of the association between this haplotype and asthma.

Example 9 Preparation of Antibody Compositions to the 127-Ile Variant ofFLAP

Substantially pure protein or polypeptide is isolated from transfectedor transformed cells containing an expression vector encoding the FLAPprotein or a portion thereof. The concentration of protein in the finalpreparation is adjusted, for example, by concentration on an Amiconfilter device, to the level of a few micrograms/ml. Monoclonal orpolyclonal antibody to the protein can then be prepared as follows:

A. Monoclonal Antibody Production by Hybridoma Fusion

Monoclonal antibody to epitopes in the FLAP protein or a portion thereofcan be prepared from murine hybridomas according to the classical methodof Kohler, G. and Milstein, C., (1975) or derivative methods thereof.Also see Harlow, E., and D. Lane. 1988.

Briefly, a mouse is repetitively inoculated with a few micrograms of theFLAP protein or a portion thereof over a period of a few weeks. Themouse is then sacrificed, and the antibody producing cells of the spleenisolated. The spleen cells are fused by means of polyethylene glycolwith mouse myeloma cells, and the excess unfused cells destroyed bygrowth of the system on selective media comprising aminopterin (HATmedia). The successfully fused cells are diluted and aliquots of thedilution placed in wells of a microtiter plate where growth of theculture is continued. Antibody-producing clones are identified bydetection of antibody in the supernatant fluid of the wells byimmunoassay procedures, such as ELISA, as originally described byEngvall, E (1980), and derivative methods thereof. Selected positiveclones can be expanded and their monoclonal antibody product harvestedfor use. Detailed procedures for monoclonal antibody production aredescribed in Davis, L. et al. Basic Methods in Molecular BiologyElsevier, New York. Section 21-2.

B. Polyclonal Antibody Production by Immunization

Polyclonal antiserum containing antibodies to heterogeneous epitopes inthe FLAP protein or a portion thereof can be prepared by immunizingsuitable non-human animal with the FLAP protein or a portion thereof,which can be unmodified or modified to enhance immunogenicity. Asuitable non-human animal is preferably a non-human mammal is selected,usually a mouse, rat, rabbit, goat, or horse. Alternatively, a crudepreparation which has been enriched for FLAP concentration can be usedto generate antibodies. Such proteins, fragments or preparations areintroduced into the non-human mammal in the presence of an appropriateadjuvant (e.g. aluminum hydroxide, RIBI, etc.) which is known in theart. In addition the protein, fragment or preparation can be pretreatedwith an agent which will increase antigenicity, such agents are known inthe art and include, for example, methylated bovine serum albumin(mBSA), bovine serum albumin (BSA), Hepatitis B surface antigen, andkeyhole limpet hemocyanin (KLH). Serum from the immunized animal iscollected, treated and tested according to known procedures. If theserum contains polyclonal antibodies to undesired epitopes, thepolyclonal antibodies can be purified by immunoaffinity chromatography.

Effective polyclonal antibody production is affected by many factorsrelated both to the antigen and the host species. Also, host animalsvary in response to site of inoculations and dose, with both inadequateor excessive doses of antigen resulting in low titer antisera. Smalldoses (ng level) of antigen administered at multiple intradermal sitesappears to be most reliable. Techniques for producing and processingpolyclonal antisera are known in the art, see for example, Mayer andWalker (1987). An effective immunization protocol for rabbits can befound in Vaitukaitis, J. et al. (1971).

Booster injections can be given at regular intervals, and antiserumharvested when antibody titer thereof, as determinedsemi-quantitatively, for example, by double immunodiffusion in agaragainst known concentrations of the antigen, begins to fall. See, forexample, Ouchterlony, O. et al., (1973). Plateau concentration ofantibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12μM). Affinity of the antisera for the antigen is determined by preparingcompetitive binding curves, as described, for example, by Fisher, D.(1980).

Antibody preparations prepared according to either the monoclonal or thepolyclonal protocol are useful in quantitative immunoassays whichdetermine concentrations of antigen-bearing substances in biologicalsamples; they are also used semi-quantitatively or qualitatively toidentify the presence of antigen in a biological sample. The antibodiesmay also be used in therapeutic compositions for killing cellsexpressing the protein or reducing the levels of the protein in thebody.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein by the one skilled in the art without departing from the spiritand scope of the invention.

REFERENCES

The following references are cited herein and are incorporated herein byreference in their entireties:

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1. A method of genotyping comprising: a) determining the identity of anucleotide at a FLAP-related biallelic marker of SEQ ID NO: 1 or thecomplement thereof in a biological sample, and b) identifying a T atsaid FLAP-related biallelic marker of SEQ ID NO: 1 wherein saidFLAP-related biallelic marker is located at position 28368 of SEQ IDNO:
 1. 2. The method according to claim 1, wherein said biologicalsample is derived from a single subject.
 3. The method according toclaim 2, wherein the identity of the nucleotides at said biallelicmarker is determined for both copies of said biallelic marker present insaid individual's genome.
 4. A method of estimating the frequency of ahaplotype for a set of biallelic markers in a control or in an asthmapositive population, comprising: a) genotyping at least one FLAP-relatedbiallelic marker according to claim 3 for each individual in saidpopulation; b) genotyping a second biallelic marker by determining theidentity of the nucleotides at said second biallelic marker for bothcopies of said second biallelic marker present in the genome of eachindividual in said population; and c) applying a haplotype determinationmethod to the identities of the nucleotides determined in steps a) andb) to obtain an estimate of said frequency.
 5. The method according toclaim 4, wherein said haplotype determination method is selected fromthe group consisting of asymmetric PCR amplification, double PCRamplification of specific alleles, the Clark algorithm, or anexpectation-maximization algorithm.
 6. A method of detecting anassociation between a haplotype and an asthma trait, comprising thesteps of: a) estimating the frequency of at least one haplotype in aasthma positive population according to the method of claim 4; b)estimating the frequency of said haplotype in a control populationaccording to the method of claim 4; and c) determining whether astatistically significant association exists between said haplotype andsaid asthma trait.
 7. The method according to claim 6, wherein saidcontrol population is an asthma negative population.
 8. The methodaccording to claim 6, wherein said control population is a randompopulation.
 9. The method according to claim 1, wherein said biologicalsample is derived from multiple subjects.
 10. The method according toclaim 1, further comprising amplifying a portion of said sequencecomprising the biallelic marker prior to said determining step.
 11. Themethod according to claim 10, wherein said amplifying is performed byPCR.
 12. The method according to claim 1, wherein said determining isperformed by an assay selected from the group consisting of ahybridization assay, a sequencing assay, a microsequencing assay and anenzyme-based mismatch detection assay.
 13. A method of estimating thefrequency of an allele of a FLAP-related biallelic marker in a controlor in an asthma positive population comprising: a) genotypingindividuals from said population for said biallelic marker according toclaim 8; and b) determining the proportional representation of saidbiallelic marker in said population.
 14. A method of detecting whetheran association exists between a genotype and a phenotype, comprising thesteps of: a) determining the frequency of at least one FLAP-relatedbiallelic marker in an asthma positive population according to themethod of claim 13; b) determining the frequency of at least oneFLAP-related biallelic marker in a control population according to themethod of claim 13; and c) determining whether a statisticallysignificant association exists between said genotype and said phenotype.15. The method according to claim 14, wherein said genotyping steps a)and b) are performed on a single pooled biological sample derived fromeach of said populations.
 16. The method according to claim 14, whereinsaid genotyping steps a) and b) are performed separately on biologicalsamples derived from each individual in said populations.
 17. The methodaccording to claim 14, wherein said control population is an asthmanegative population.
 18. The method according to claim 14, wherein saidcontrol population is a random population.
 19. A method of determiningwhether a human is at an increased risk of developing asthma,comprising: a) genotyping a first FLAP-related marker according to claim8, b) determining the identity of at least a second FLAP relatedbiallelic marker of SEQ ID NO: 1, said second nucleotide being selectedfrom: a C at position 4670, a C at position 16288, an A at position16347, a T at position 16440, a G at position 28336, a C at position42445 or a G at position 38681, and c) identifying the human as havingan increased risk of developing asthma when i) a T is identified atposition 28368 of SEQ ID NO: 1 and (ii) a C at position 4670, a C atposition 16288, an A at position 16347, a T at position 16440, a G atposition 28336, a C at position 42445 or a G at position 38681 isidentified at the second FLAP related biallelic marker.
 20. The methodaccording to claim 19, wherein the identity of the nucleotides at thefollowing combinations of biallelic markers in SEQ ID NO: 1 isdetermined: nucleotides 28368 and 16440; nucleotides 28368 and 28336;nucleotides 28368 and 16288; nucleotides 28368 and 38681; nucleotides28368, 16347 and 42445; nucleotides 28368, 16347 and 38681; nucleotides28368, 16347 and 28336; nucleotides 28368, 16347, 42445 and 4670;nucleotides 28368, 16347, 42445, 38681 and 28336; nucleotides 28368,16347, 42445, 38681 and 4670; nucleotides 28368, 16347, 42445, 38681,28336 and 4670; or nucleotides 28368, 16347, 42445, 38681, 28336, 4670and
 16440. 21. The method according to claim 20, wherein the identity ofthe nucleotides at the following combination of biallelic markers in SEQID NO: 1 is determined: nucleotides 28368 and
 16440. 22. The methodaccording to claim 20, wherein the identity of the nucleotides at thefollowing combination of biallelic markers in SEQ ID NO: 1 isdetermined: nucleotides 28368 and
 28336. 23. The method according toclaim 20, wherein the identity of the nucleotides at the followingcombination of biallelic markers in SEQ ID NO: 1 is determined:nucleotides 28368 and
 16288. 24. The method according to claim 20,wherein the identity of the nucleotides at the following combination ofbiallelic markers in SEQ ID NO: 1 is determined: nucleotides 28368 and38681.
 25. The method according to claim 20, wherein the identity of thenucleotides at the following combination of biallelic markers in SEQ IDNO: 1 is determined: nucleotides 28368, 16347 and
 42445. 26. The methodaccording to claim 20, wherein the identity of the nucleotides at thefollowing combination of biallelic markers in SEQ ID NO: 1 isdetermined: nucleotides 28368, 16347 and
 38681. 27. The method accordingto claim 20, wherein the identity of the nucleotides at the followingcombination of biallelic markers in SEQ ID NO: 1 is determined:nucleotides 28368, 16347 and
 28336. 28. The method according to claim20, wherein the identity of the nucleotides at the following combinationof biallelic markers in SEQ ID NO: 1 is determined: nucleotides 28368,16347, 42445 and
 4670. 29. The method according to claim 20, wherein theidentity of the nucleotides at the following combination of biallelicmarkers in SEQ ID NO: 1 is determined: nucleotides 28368, 16347, 42445,38681 and
 28336. 30. The method according to claim 20, wherein theidentity of the nucleotides at the following combination of biallelicmarkers in SEQ ID NO: 1 is determined: nucleotides 28368, 16347, 42445,38681 and
 4670. 31. The method according to claim 20, wherein theidentity of the nucleotides at the following combination of biallelicmarkers in SEQ ID NO: 1 is determined: nucleotides 28368, 16347, 42445,38681, 28336 and
 4670. 32. The method according to claim 20, wherein theidentity of the nucleotides at the following combination of biallelicmarkers in SEQ ID NO: 1 is determined: nucleotides 28368, 16347, 42445,38681, 28336, 4670 and
 16440. 33. The method according to claim 1,further comprising determining the identity of at least one additionalnucleotide at a FLAP-related biallelic marker of SEQ ID NO: 1, said atleast one additional nucleotide being selected from: a C at position4670, a C at position 16288, an A at position 16347, a Tat position16440, a G at position 28336, a C at position 42445 or a G at position38681.
 34. The method according to claim 33, wherein the identity of thenucleotides at the following combinations of biallelic markers in SEQ IDNO: 1 is determined: nucleotides 28368 and 16440; nucleotides 28368 and28336; nucleotides 28368 and 16288; nucleotides 28368 and 38681;nucleotides 28368, 16347 and 42445; nucleotides 28368, 16347 and 38681;nucleotides 28368, 16347 and 28336; nucleotides 28368, 16347, 42445 and4670; nucleotides 28368, 16347, 42445, 38681 and 28336; nucleotides28368, 16347, 42445, 38681 and 4670; nucleotides 28368, 16347, 42445,38681, 28336 and 4670; or nucleotides 28368, 16347, 42445, 38681, 28336,4670 and
 16440. 35. The method according to claim 33, wherein theidentity of the nucleotides at the following combination of biallelicmarkers in SEQ ID NO: 1 is determined: nucleotides 28368 and
 16440. 36.The method according to claim 33, wherein the identity of thenucleotides at the following combination of biallelic markers in SEQ IDNO: 1 is determined: nucleotides 28368 and
 28336. 37. The methodaccording to claim 33, wherein the identity of the nucleotides at thefollowing combination of biallelic markers in SEQ ID NO: 1 isdetermined: nucleotides 28368 and
 16288. 38. The method according toclaim 33, wherein the identity of the nucleotides at the followingcombination of biallelic markers in SEQ ID NO: 1 is determined:nucleotides 28368 and
 38681. 39. The method according to claim 33,wherein the identity of the nucleotides at the following combination ofbiallelic markers in SEQ ID NO: 1 is determined: nucleotides 28368,16347 and
 42445. 40. The method according to claim 33, wherein theidentity of the nucleotides at the following combination of biallelicmarkers in SEQ ID NO: 1 is determined: nucleotides 28368, 16347 and38681.
 41. The method according to claim 33, wherein the identity of thenucleotides at the following combination of biallelic markers in SEQ IDNO: 1 is determined: nucleotides 28368, 16347 and
 28336. 42. The methodaccording to claim 33, wherein the identity of the nucleotides at thefollowing combination of biallelic markers in SEQ ID NO: 1 isdetermined: nucleotides 28368, 16347, 42445 and
 4670. 43. The methodaccording to claim 33, wherein the identity of the nucleotides at thefollowing combination of biallelic markers in SEQ ID NO: 1 isdetermined: nucleotides 28368, 16347, 42445, 38681 and
 28336. 44. Themethod according to claim 33, wherein the identity of the nucleotides atthe following combination of biallelic markers in SEQ ID NO: 1 isdetermined: nucleotides 28368, 16347, 42445, 38681 and
 4670. 45. Themethod according to claim 33, wherein the identity of the nucleotides atthe following combination of biallelic markers in SEQ ID NO: 1 isdetermined: nucleotides 28368, 16347, 42445, 38681, 28336 and
 4670. 46.The method according to claim 33, wherein the identity of thenucleotides at the following combination of biallelic markers in SEQ IDNO: 1 is determined: nucleotides 28368, 16347, 42445, 38681, 28336, 4670and 16440.